Interleukin-2 restores the depressed allogeneic cell-mediated lympholysis and natural killer cell activity in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by a variety of profound T-cell abnormalities among which are decreased cytotoxic capacity measured by allogeneic cell-mediated lympholysis (CML), natural killer cell (NK) activity, and decreased lymphokine production. In a group of 13 patients with active SLE, allogeneic CML, tested by a 4-hr 51Cr-release assay, was 18.2 +/- 2.7% while in the group of normal individuals CML was 41.2 +/- 2.7%. If optimal doses of affinity-purified interleukin-2 (IL-2) were present during the mixed lymphocyte culture, the CML of SLE patients was increased to normal levels (40.4 +/- 4.0%). In contrast, interferon-alpha (IFN-alpha) increased (but not significantly) the levels of CML. Mixed lymphocyte reaction, tested by tritiated thymidine incorporation, was also decreased in the group of patients (14,820 +/- 815 cpm vs 28,972 +/- 5880 cpm in normals) and it was increased to normal levels if IL-2, but not IFN-alpha was added to the cultures. NK activity was decreased in the group of patients tested by 51Cr-release assay, harvested at 6 and 18 hr. IL-2 increased the NK activity up to normal levels, while IFN-alpha was only partially efficacious. These results demonstrate that IL-2, but not IFN-alpha, can potentiate or even fully restore the deficient cytotoxic effector function of peripheral mononuclear cells in patients with SLE.     

Natural killer cells and interferon responses in patients with systemic lupus erythematosus.

Natural killer (NK) cell activity was studied in 23 patients with systemic lupus erythematosus (SLE). The overall NK activity was lower in patients with SLE than in normal female individuals. Patients with clinically active SLE disease had slightly lower NK activity than the patients with inactive disease. Other clinical parameters as well as treatment status did not correlate with NK activity. Interferon (IFN) enhanced the NK activity of normal individuals and of 11 SLE patients, while it did not enhance in the remaining 12 patients. The patients whose NK activity was enhanced by beta-IFN had significantly higher initial activity than those who did not respond to beta-IFN. Furthermore, peripheral mononuclear cells (MNC) from IFN responders produced gamma-IFN after stimulation with concanavalin A (Con A) in titres comparable to those of normals. In contrast, peripheral MNC from beta-IFN non-responders failed to produce significant titres of gamma-IFN after stimulation with Con A. These results indicate that certain patients with SLE have low NK activity, which is generally paralleled by an inability to respond to exogenous beta-IFN and by blunted production of gamma-IFN after stimulation with Con A.     

Decreased natural and antibody-dependent cellular cytotoxic activities in intravenous drug abusers

Peripheral blood lymphocytes from 14 adult male patients admitted to the hospital with complications of intravenous drug abuse (IDA) were examined for natural killer (NK) and antibody-dependent cellular cytotoxic (ADCC) activities, lectin-dependent cellular cytotoxicity, and interferon (IFN)- and interleukin 2 (IL-2)-induced NK activity. Serum was also assayed for circulating interferon levels and soluble factor(s) capable of suppressing the cytotoxic potential of allogeneic lymphocytes from healthy donors. IDA patients demonstrated significantly decreased levels of NK and ADCC activities compared to age- and sex-matched healthy controls. The lectin, phytohemagglutinin, could significantly enhance the cytotoxicity of IDA lymphocytes; however, activity was not completely restored to normal levels. IDA sera demonstrated a significant inhibitory effect on the NK and ADCC activities of normal allogeneic lymphocytes, and these sera contained negligible levels of circulating IFN. Although the NK activity of IDA lymphocytes could not be restored completely to normal levels by either IFN-alpha or IL-2, the percentage enhancement of cytotoxicity was remarkably higher in IDA patients with significantly reduced NK activity than that observed using PBL from patients with near normal NK activity. The ability of IFN or IL-2 to enhance the decreased cytotoxicity of PBL from drug abusers suggests a novel therapeutic approach to the management of the complications of IDA.     

Further studies of natural killer cell function in Chediak-Higashi patients.

Spontaneous natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of blood lymphocytes against five human tumour cell lines (K562, Molt-4, HL-60, Chang, Daudi) and three mouse tumour lines (YAC, P815, RBL-5) were ten- to 100-fold lower than normal in six patients with Chediak-Higashi (CH) disease. NK and ADCC were defective at 4 hr, and less so at 18 hr. The NK activity in normals and CH patients was mediated in part by FcR+, E- effector cells. ADCC against human erythrocytes was normal in CH patients, as were lectin-dependent cytolysis and mixed lymphocyte proliferative responses. Phagocytosis of antibody-coated ox erythrocytes was normal in CH patients as well. These observations confirm that the CH syndrome is associated with a profound and selective defect in NK and ADCC activity against tumour cells, whereas other mononuclear cell-mediated functions are normal.     

Natural killer cell function in trisomy-21 (Down''s syndrome)

Natural killer (NK) activity and antibody-dependent cell mediated cytotoxicity (ADCC) against a human myeloid target cell line (K 562) was measured in adult patients with trisomy-21 (Down's syndrome) and in chromosomally normal age and sex matched control subjects. The effect of human leucocyte interferon (IFN-alpha) on the NK activity was also estimated. Spontaneous NK activity was stronger in the adult patients with trisomy-21 than in the healthy controls, but the difference did not reach statistical significance. The augmentation of NK activity by IFN-alpha, measured using lymphocytes not depleted of monocytes as effector cells, was statistically significant in both the trisomic patients (P less than 0.004) and the healthy controls (P less than 0.0005). Using monocyte and macrophage depleted lymphocytes in the patients with trisomy-21 the NK activity proved stronger than in the healthy controls, but not significantly and IFN-alpha did not augment it as it did in the healthy controls (P = n.s., P less than 0.05), for augmentations respectively). These results support the view that monocytes and macrophages are connected with the NK cell system. ADCC correlated with NK activity in both groups. Since NK cells are important components of many immune processes, including tumour and virus and/or bacteria-infected cell elimination, and have regulatory functions in immune reactions, the deficient augmentation of trisomic NK cells shown in vitro with extrinsic human leucocyte interferon may, paradoxically be an explanation for the greater susceptibility of trisomic individuals to lymphatic leukaemia and virus and bacterial infections. In vivo, this could be explained by the more potent secondary suppression by the 'immune' interferon produced by the virus, bacteria and malignant cells. In other words, the potential of the 'fighting couple' of the immune system, NK cell/interferon, is perhaps disturbed genetically due to the chromosome 21.     

Endogenous and interferon-augmented natural killer cell activity of human peripheral blood mononuclear cells in vitro. Studies of patients with multiple sclerosis, systemic lupus erythematosus or rheumatoid arthritis.

Peripheral blood mononuclear cells (PBMC) of normal human donors are spontaneously cytotoxic for certain tumour-derived and virus-infected target cells. This so-called natural killing (NK) can be augmented by the action of interferons (IFN) and by IFN-inducers. In this study, we have compared both endogenous and augmented NK activity of normal donors with that of patients suffering from either multiple sclerosis (MS), systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). Endogenous NK was assayed using an NK susceptible target cell (K562), and augmented NK using a target cell (WI-L2) which is lysed only by NK effector cells that have been pre-stimulated by IFN or IFN-inducers. While NK function appeared normal in RA patients, this study confirms previous reports of defective endogenous NK in many MS and SLE patients. In addition, anomalous IFN-augmented NK was also detected in many patients with these two diseases, indicating that defective NK function cannot always be corrected by IFN treatment in vitro. Analysis of IFN production, endogenous NK and IFN-augmented NK by individual patients with MS or SLE showed the defects in their IFN-NK systems to be highly selective, suggesting that individual components of this system may operate independently.     

Effects of interferon-alpha on the expression of a lupus idiotype in normal humans

Interferon (IFN) has extensive immunoregulatory effects but its role in systemic lupus erythematosus (SLE) remains obscure. The observations that a high proportion of patients with active SLE have increased IFN levels in their sera, and that IFN injected to lupus-prone mice aggrevates their disease, led us to examine the effects of IFN on the production of 16/6--a high frequency idiotype of monoclonal anti-DNA antibodies produced by human-human hybridomas derived from SLE patients. Peripheral blood mononuclear cells (PBMC) of healthy donors or of patients with SLE were incubated with IFN and pokeweed mitogen (PWM). Seven-day supernatants were assayed for total IgM, for IgM with 16/6 idiotype, and for IgM anti-DNA activity. PWM-stimulated PBMC of all healthy donors examined produced the 16/6 idiotype (mean 2.5 ng/ml). A significant increase of 16/6 in normals (above the level with PWM alone) was noted with 10-100 u/ml of IFN-alpha but not with 500 u/ml. In 3/10 normals the addition of IFN-alpha resulted in detectable anti-DNA activity. The IFN-induced increase in 16/6 idiotype was significantly more than the increase in IgM (335% vs 47% above baseline, with 10 u/ml of IFN). These effects of IFN could not be demonstrated in the absence of PWM nor in T-cell-depleted preparations. Recombinant IFN-gamma had no augmenting effect on 16/6 production. Three SLE patients in remission had elevated levels of 16/6 in their PBMC supernatant (15-200 ng/ml) which could not be further augmented by IFN. Thus, we have demonstrated the potential of PWM-stimulated normal lymphocytes to generate a "lupus" idiotype and shown that production of this idiotype requires T cells and is preferentially enhanced by IFN-alpha. Further studies of the effects of IFN on the expression of anti-DNA antibodies may clarify a postulated role of IFN in autoimmune diseases.     

Interferon and natural killer cells in systemic lupus erythematosus.

Natural killer (NK) cell activity against several types of target cells was found to be subnormal in patients with systemic lupus erythematosus (SLE). Interferon (IFN) boosted the NK activity of cells from SLE patients to a significantly lesser degree than cells from normal controls. The production of IFN after stimulation of blood cells with Sendai virus was significantly decreased in SLE patients with active disease, and in a substantial proportion of the patients the production of phytohaemagglutinin (PHA)-induced IFN was below normal limits. Although the production of virus-induced IFN was clearly inversely correlated to disease activity no such correlation was observed for PHA-induced IFN. Serum levels of both pH2 stable and pH2 labile IFN were significantly higher in SLE patients than in controls. The findings clearly show that SLE is associated with abnormalities in the NK cell-IFN system but it cannot be stated whether these abnormalities are causally related to the development of disease or are secondary to pathological changes in SLE.     

Immune status in crohn's disease

Summary In Crohn's disease (CD) a potential etiopathogenetic role of the antibody dependent cell mediated cytotoxicity (ADCC) has been discussed. By means of a⁵¹Cr release assay with an established human lymphoblastoid cell line as target, the in vitro ADCC activity was studied in the peripheral blood of 34 patients with CD. 11 patients with inflammatory bowel disease other than CD or ulcerative colitis (group D) and 45 sex and age matched healthy people served as controls.In unseparated mononuclear cell suspensions, we found a significantly lower ADCC activity in CD patients and in group D than in normals. Depletion of both phagocytic and plastic adherent cells, i.e. macrophages, resulted in complete normalisation of the ADCC activity in all patients of group D and also two-thirds of the CD patients. However, the remaining one third CD patients still exhibited ADCC activity below 1 standard deviation of normal. Most of these latter CD patients had highly active disease.Thus, this study showes that the general ADCC per se is normal in CD, except for a decrease in patients with high activity of disease. However, the normal general ADCC activity per se is significantly suppressed by phagocytic, plastic adherent cells. In this regard similar results were found in group D.Parts of this work were presented at the 8th Leukocyte Culture Workshop, Berlin     

Natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities can be differentiated by their different sensitivities to interferon and prostaglandin E1

Though purported to be identical cells (or in identical populations of cells), the natural killer (NK) cell mediating spontaneous natural cytotoxicity and the killer (K) cell mediating antibody-dependent cellular cytotoxicity (ADCC) may not be totally identical, at least in susceptibility to regulation by the immunomodulators prostaglandin E₁ (PGE₁) and interferon (IFN). We demonstrate here that NK cells are always enhanced by IFN, while K cells are inhibited from binding targets, resulting in fewer effectors at optimal concentrations of antibody. Only at 10- to 100-fold suboptimal concentrations of antibody is ADCC activity enhanced. As measured by magnitude of inhibition and dose-response titration, ADCC activity is less sensitive to the effects of PGE₁ than is NK activity in the⁵¹Cr release assay and single-cell assay. After overnight incubation with or without PGE₁, whatever sensitivity ADCC activity had to PGE₁ is lost. However, NK cells incubated in the presence of PGE₁ overnight are still sensitive to inhibition. Indomethacin boosts NK activity without having any effect on ADCC activity. Finally, NK activity is substantially reduced by overnight incubation of cells at room temperature, which has no effect on K cells.     

Spontaneous and interferon-induced activity of natural killer cells in patients with liver cirrhosis and chronic active hepatitis

We investigated spontaneous and gamma-interferon stimulated natural killer (NK) cell activity and antibody-dependent cellular cytotoxicity (ADCC) in patients with chronic liver disease: A significantly reduced NK activity was found in alcoholic liver cirrhosis (LZ) and in chronic active hepatitis (CAH), as compared to healthy control individuals (p less than 0.001 and p less than 0.005, respectively). Patients with primary biliary cirrhosis (PBC) or with alcoholic non-cirrhotic liver disease had normal NK activities. Furthermore, ADCC was found to be significantly reduced in patients with CAH (p less than 0.05). The addition of gamma-interferon to peripheral blood mononuclear cells in vitro induced a similar increase in NK cell activity in all studied groups of patients. The reduced NK cell activity found in patients with LZ and CAH could constitute an important mechanism in the pathogenesis and contribute to the high incidence of virus-associated hepatic malignancies in these disorders.     

Interferon production of vitro by leucocytes from patients with systemic lupus erythematosus and rheumatoid arthritis.

The production of interferons (IFN) by peripheral blood leucocytes from normal donors an patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) has been investigated in response to several IFN inducers in vitro. Whereas IFN responses of RA donors did not differ significantly from the normal group, those of SLE patients were significantly reduced, and many of these patients failed to respond to all. Patients with active or acute SLE responded significantly less well than those with inactive disease. There was no apparent effect of steroid therapy on the IFN responses of either SLE or RA patients. These data may indicate a basic immunological defect of the circulating leucocytes of SLE patients, which may be responsible for some of the in vitro lymphocyte anomalies reported for this disease.     

Immunoradiometric assay of a recombinant human alpha-2 interferon (SCH 30500).

A sensitive immunoradiometric assay for recombinant alpha-2 interferon (IFN-alpha 2; SCH 30500) was developed by using the monoclonal antibody NK2. A polystyrene bead with polyclonal sheep anti-human IFN-alpha (Hu IFN-alpha) antibody adsorbed to it formed the solid phase of the assay. The monoclonal antibody, labeled with 125I, reacted with the IFN-alpha 2 which bound to the polyclonal antibody on the bead. The assay was sensitive, capable of measuring less than 5 IU/ml, and it was precise. Coefficients of variation were less than 10%, often less than 5%. Interassay coefficients of variation for the same sample were also less than 10%. Specificity of the monoclonal antibody for IFN-alpha 2 was greater than for Hu IFN-alpha produced by leukocytes. In specificity studies it was necessary to add at least 200 IU of Hu IFN-alpha per ml to 25 IU of IFN-alpha 2 per ml before the Hu IFN-alpha affected the accuracy of the assay of IFN-alpha 2. Accuracy as determined by correlation studies with the antiviral cytopathic effect assay was very high. Results of the two assays usually agreed within 15%. This correlation was maintained in studies of IFN-alpha 2 after storage under accelerated degradation conditions, when the IFN was cleaved into smaller sequences of amino acids by cyanogen bromide and when the IFN was carboxymethylated.     

Abnormal macrophages and NK cell cytotoxicity in human systemic lupus erythematosus and the role of interferon and serum factors

Macrophage (MO) and natural killer (NK) cell mediated cytotoxicity to K562 target cells were strikingly decreased in patients with systemic lupus erythematosus (SLE). SLE NK cells failed to release soluble factor(s) for lysing the targets. IFN-induced enhancement of both types of cytotoxicity was impaired. NK cells from healthy subjects kept their activity in culture with or without IFN for more than six days whereas SLE NK cell activity declined to zero at day 3. So, the increased IFN level of many SLE patients and a possible prior IFN priming effect seemed unrelated to the insensitivity to exogenous IFN in vitro. Inhibition factor(s) of SLE serum suppressed NK cytotoxicity in the presence of IFN whereas IFN sensitivity of MO remained unaffected indicating the complex regulation by serum components of immune reactions.     

Interferons in rheumatoid arthritis: alterations in production and response related to disease activity

Various markers associated with the production of and response to interferons (IFNs) were studied in patients with either inactive rheumatoid arthritis (RA) or active RA, and in healthy subjects. The IFN markers assessed were serum and synovial fluid (SF) levels, the activity in peripheral blood leukocytes (PBL) of (2'-5') oligoadenylate synthetase (OAS), and the production in vitro by PBL of IFN-alpha/beta in response to Sendai virus or Poly(I):Poly(C) as inducers, and of IFN-gamma using PHA or Con A as inducers. IFN activity, tested by antiviral assays using two different cell lines, was not demonstrable in the serum of any patient with RA. The activity of (2'-5') OAS in PBL, which may indirectly indicate exposure of leukocytes to IFN, was increased in RA compared with healthy subjects, more so in patients with inactive RA. The production of IFN-alpha/beta by PBL in response to Sendai virus was low in active RA but high in inactive RA, relative to production in healthy subjects. The production of IFN-gamma by PBL in RA was lower than in healthy subjects, more so in active RA. Thus inactive RA (remission status) is marked by evidence of PBL having been influenced by interferon and being a state of augmented inducibility to an IFN-alpha/beta stimulus, whereas active RA is associated with low inducibility of PBL to an IFN stimulus, but no evidence of IFN production in vivo. Our findings underscore the relevance of interferon to remission/activity in rheumatoid arthritis.     

Frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus

The frequency of anti-DNA antibody producing cells from normals and patients with systemic lupus erythematosus (SLE) was determined. Peripheral blood lymphocytes (PBL) from normals and patients with SLE were cultured for 8 and 15 days with and without transformation by Epstein-Barr virus (EBV). Culture supernatants were examined for the presence of anti-DNA antibody using an enzyme-linked immunosorbent assay. We found that PBL from patients with SLE spontaneously produce anti-DNA antibodies whereas PBL from normals do not. After EBV transformation, anti-DNA antibody producing cells were detected in both cultures from patients with SLE as well as from normals. These data suggest that the high levels of anti-DNA antibody observed in patients with SLE represent activation of B cells committed to anti-DNA antibody production and that such cells are present but are not activated in normal individuals.     

Cytotoxic activity of peripheral blood and cerebrospinal fluid lymphocytes from patients with multiple sclerosis and other neurological diseases: analysis at the single cell level of the relationship of cytotoxic effectors and interferon-producing cells

The production of interferon (IFN alpha) in relationship to NK and ADCC activity of peripheral blood and cerebrospinal lymphocytes was examined at the single cell level in patients with multiple sclerosis (MS) and other neurological diseases (OND) compared with age- and sex-matched controls. IFN-producing cells were assessed by indirect immunofluorescent scoring of cytoplasmic IFN+ cells. Peak production of cytoplasmic IFN alpha in nylon wool-passed ( NWP ) cells occurred between 5 and 17 hr in vitro under the inductive stimulus of MOLT 4, K562, or antibody-coated Chang liver cells. The proportion of K562- and MOLT 4-induced IFN alpha-positive cells in the total lymphocyte and target-binding cell (TBC) population was significantly lower in MS NWP -peripheral blood lymphocytes (PBL) than in OND and normal controls; this was in direct relationship to a decreased percentage of NK cells in MS PBL. In contrast MS cells responded the same as controls (total IFN+ cells) or higher than controls (IFN+-TBC) after IFN alpha induction by antibody-coated Chang, the ADCC target, in parallel with elevated ADCC activity by MS PBL. MS CSF contained a higher proportion of total IFN+ cells but a similar proportion of IFN+-TBC as their homologous NWP PBL population. In OND CSF, both the percentage of total IFN+ and the percentage of IFN+-TBC were higher than in OND blood and higher than their respective MS CSF populations. The relationship of IFN-producing cells in the central nervous system (CNS) to putative cytotoxic cells is discussed.     

Cell-mediated immunity and biological response modifiers in insulin-dependent diabetes mellitus complicated by end-stage renal disease

Previous studies have shown several immunoregulatory abnormalities in insulin-dependent diabetes mellitus (IDDM). In this report we compared peripheral blood mononuclear cells (PBMC) from patients with IDDM complicated by end-stage renal disease (ESRD) to those from normal subjects and from patients with ESRD of different etiologies for their: natural killer (NK) and antibody-dependent cell-mediated cytotoxic (ADCC) activities; modulation of NK and ADCC activities by biological response modifiers (BRM) including purified human lymphoblastoid interferon, human recombinant alpha-2 interferon, human gamma interferon and human recombinant interleukin 2; proliferative response of T and B lymphocytes to concanavalin A (Con A), phytohemagglutinin and pokeweed mitogen, and ability to produce T-cell growth factor (interleukin 2; IL-2). PBMC of diabetic patients demonstrated significantly lower NK activity than normal and ESRD subjects. Upon treatment with BRM, NK activity was augmented and achieved normal levels. ADCC activity was not different from that of normal controls and exhibited similar increases when stimulated by BRM. The proliferative responses to Con A, phytohemagglutinin and pokeweed mitogen as well as IL-2 production in response to Con A stimulation were significantly lower in the IDDM group. Our results indicated that NK cells from patients with IDDM can respond to IL-2 with enhanced cytotoxicity, and, because activation of resting T cells by mitogenic stimuli depends on the production of IL-2 as well as the appearance of a receptor for IL-2, our finding of low levels of in vitro IL-2 production by PBMC from patients with IDDM may explain the depressed NK activity and the observed poor response to T-cell mitogens.     

Antibody-dependent cellular cytotoxicity of mononuclear cells from asthmatics tested in three in vitro assays.

Using the chicken red blood cell assay, the human Chang liver cell assay, and a lymphoblastoid cell assay, mononuclear cells from asthmatics and normals were tested for antibody-dependent cellular cytotoxicity (ADCC) capacity. Mononuclear cell preparations from perennial nonallergic asthmatics with a history of asthma associated with viral infections had a reduced ADCC capacity in the chicken red blood cell assay, an increased ADCC capacity in the Chang liver cell assay, and normal ADCC capacity in the lymphoblastoid cell assay. The data also suggested that perennial nonallergic asthmatics had increased percentages of surface IgG-negative lymphocytes in the peripheral blood when compared to normals.     

Immunological and Functional Characteristics of Peripheral Blood and Synovial Fluid Lymphocytes from Patients with Rheumatoid Arthritis

Spontaneous (SCMC) and antibody dependent cellular cytotoxicity (ADCC); mitogenic responsiveness (PHA, Con A, PPD, dextran and pokeweed) as well as lymphocyte subpopulations (E-, EA-, EAC-rosettes, S-Ig) were studied simultaneously in peripheral blood (PBL) and synovial fluid lymphocytes (SFL) of fifteen patients with rheumatoid arthritis. Marked differences were observed in the cytotoxic activity of SFL and PBL. Whereas SCMC activity of SFL was always significantly elevated above the cytotoxic levels of PBL, the reverse was true for the ADCC reaction; here, 50% of the patients showed a decreased cytotoxicity of SFL compared to PBL. Synovial fluid neutrophils (SFN) were found to be inactive in both cytotoxic assays. No differences were found in ADCC activity of PBL between normal controls and RA patients. In SCMC assays a significantly increased activity of control PBL was only observed at L/T ratios of 100:1. Overnight incubation of PBL from RA patients and normal controls resulted in a marked decrease in SCMC and, to a smaller extent, in ADCC activity. SFL from three out of four patients lost less SCMC activity after overnight incubation than the corresponding PBL. In one patient even an increased activity in both cytotoxic systems was obtained. Regarding lymphocyte populations, T-cells were significantly decreased in PBL of RA patients. With the exception of a significantly lowered percentage of C3 receptor positive cells in SFL, no significant differences were recorded in the lymphocyte distribution between the patients' PBL and SFL. In the RA patients, the response to T-cell mitogens was significantly depressed in SFL while PPD and pokeweed reactivity was equal to that of PBL.