Lymphocytotoxic antibodies in systemic lupus erythematosus: evidence for reactivity with i antigen.

Sera from ten patients with systemic lupus erythematosus (SLE) were tested for lymphocytotoxic antibodies (LCA) before and after absorption with cord and adult erythrocytes at 4 degrees C. Seven of the sera showed a significant reduction in cytotoxicity after absorption with the cord erythrocytes, but minimal or no reduction following absorption with the adult red blood cells. Of the remaining three sera, the cytotoxicity was equally reduced by cord and adult red cells in one and was unaffected by absorption in two. Eluates were prepared from the two sera using cord and adult red blood cells. The cytotoxicity of the cord cell eluates was significantly greater than those of the adult red cell eluates. These data indicate that the LCA in most SLE sera have specificity for the i antigen, which is present on lymphocytes and cord erythrocytes.     

Evaluation of soluble tumor necrosis factor (TNF) receptors and TNF receptor antibodies in patients with systemic lupus erythematodes,progressive systemic sclerosis,and mixed connective tissue disease

Two TNF binding proteins have been characterized as soluble fragments of TNF receptors. We measured the plasma concentrations of soluble type A (p75) and type B (p55) TNF receptors in patients with systemic lupus erythematodes (SLE), progressive systemic sclerosis (PSS), and mixed connective tissue disease (MCTD). In SLE and PSS patients plasma concentrations of both types of TNF receptors and in MCTD patients type A TNF receptors were significantly elevated compared to controls. Plasma concentrations of both soluble TNF receptors were highly correlated in SLE, PSS, and MCTD patients, indicating a possible coregulation of both TNF receptors. In contrast, soluble interleukin 2 receptor (sCD 25) plasma concentrations were not correlated and seem to be an independent parameter. The soluble forms of the TNF receptors neutralize TNF in cytotoxicity assays and are functionally active as TNF antagonists. In one patient with SLE, autoantibodies against type A TNF receptors were detected, TNF, and TNF did not interfere with the autoantibody binding to the receptor.     

Antibody-dependent and phytohaemagglutinin-induced lymphocyte cytotoxicity in systemic sclerosis.

Cell-mediated cytotoxicity was examined in thirty-seven patients with systemic sclerosis using both whole blood and purified peripheral blood mononuclear cells (PBM) to measure antibody-dependent (ADCC) and phytohaemagglutinin (PHA) induced lymphocyte cytotoxicity to 51Cr-labelled Chang liver cells. In twenty-three mildly affected patients, ADCC and PHA-induced cytotoxicity did not differ from that found in control populations. By contrast, fourteen patients severely affected by extensive visceral disease showed reductions in both ADCC and PHA-induced cytotoxicity which were more marked in whole blood assays (P less than 0.001) than in those performed with PBM (P less than 0.05). The addition of patient's sera to control cytotoxicity assays suggested that blocking or suppressive serum factors could only account for some of the disproportionate reduction in whole blood cytotoxicity which, in the main, must be due to a lack of circulating effector cells. These results are in agreement with previous findings of reduced numbers of circulating thymus-dependent lymphocytes in patients with severe disease, a defect of cell-mediated immunity that may result from the chronic antigenic stimulation of an autoimmune disease process.     

Independence of depressed lectin-dependent cell-mediated cytotoxicity from interleukin 2 production in patients with systemic lupus erythematosus.

The relationship of lectin-dependent cell-mediated cytotoxicity (LDCC) to interleukin-2 (IL-2) production was studied in healthy subjects and in patients with systemic lupus erythematosus (SLE). Profoundly depressed levels of LDCC were elicited by peripheral blood mononuclear cells (PBMC) from nine patients with active SLE in comparison to LDCC from seven controls, and eleven inactive SLE donors, using 3H-TdR-prelabelled adherent HEP-2 cells as targets in a 24 h assay with 25 micrograms/ml Con A. In parallel experiments, no individual correlation was found between LDCC activity and IL-2 production for healthy or SLE subjects. Further, no major differences were detected in IL-2 release when the three groups of donors were compared, a tendency observed at the Con A doses (5 and 25 micrograms/ml) and incubation times (24, 48, and 72 h) used to induce IL-2 production. In additional studies, impaired Con A-induced blastogenesis was noted for PBMC from active SLE patients in comparison to the PBMC from the controls or patients with inactive SLE. While strong individual correlation was obtained between blastogenesis and IL-2 secretion in controls and patients with inactive SLE, no such relationship was found in patients with active SLE. While addition of exogenous IL-2 to the cytotoxicity assay considerably enhanced LDCC by healthy donors it failed to improve LDCC by patients with active SLE. These data suggest that depressed LDCC and Con A-induced blastogenesis of patients with active SLE may not be related to impaired IL-2 production but rather to an inherent dysfunction of the effector lymphocytes, including their unresponsiveness to IL-2.     

Antibody-dependent direct cytotoxicity of human lymphocytes. I. Studies on peripheral blood lymphocytes and sera of patients with systemic lupus erythematosus.

Antibody-dependent direct cytotoxicity (ADDC) is generally believed to be unrelated to T-cell function in experimental animals. The role of ADDC in humans and its clinical usefulesss was evaluated in patients with systemic lupus erythematosus (SLE) and normal controls. Peripheral blood lymphocytes from patients with active SLE were unable to lyse antibody-coated target cells in vitro to the same degree as lymphocytes from patients with inactive SLE and controls. Sera from patients with active SLE suppressed ADDC by lymphocytes derived from normal controls and this abnormality was not corrected by overnight incubation or by extensive washing of lymphocyte preparations. Although there was poor correlation between ADDC and the proportions of B cells and null cells in effector lymphocyte populations from SLE patients and controls, it is concluded that this assay provides another means of determining immune competence in man.     

Deficient spontaneous cell-mediated cytotoxicity and lectin-induced cellular cytotoxicity by peripheral blood mononuclear cells from Thai adults naturally infected with malaria.

To assess general cytotoxic effector cell capabilities by peripheral blood mononuclear cells from patients with active malaria infections, we examined antibody-dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and lectin-induced cellular cytotoxicity by using human and chicken erythrocyte, Chang cell line, and K562 cell line targets. By using human erythrocyte and Change cell line targets, we found that Thai adults naturally infected with malaria had significantly impaired lectin-induced cellular cytotoxicity. In addition, spontaneous cell-mediated cytotoxicity was deficient with K562 but not with Chang cell line targets. Finally, no change in antibody-dependent cellular cytotoxicity was observed when chicken erythrocyte or Chang cell line targets were used. These observations, coupled with our previous observations of a physical loss of peripheral blood T cells, the presence of lymphocytotoxic serum antibodies, and defective T suppressor cell generation in patients with malaria, indicate that major alterations in the cellular immune system occur in patients with active malaria infections.     

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.     

Decreased cell-mediated cytotoxicity against virus-infected cells in systemic lupus erythematosus.

Cell-mediated cytotoxicity, directed against virus-infected tissue culture cells, was studied with peripheral blood mononuclear cells from 11 patients with systemic lupus erythematosus (SLE) and 12 matched, normal subjects in a 51Cr release assay. Baseline (preimmunization) levels of cytotoxicity against target cells infected with influenza A/Victoria, influenza B/Hong Kong, Newcastle disease virus, and herpes simplex virus were significantly decreased in patients with SLE compared to normal subjects (P less than 0.001), although serum antibody levels to the respective viruses were similar in both groups. After intramuscular administration of inactivated influenza A/Victoria vaccine, SLE patients failed to generate elevated levels of cytotoxicity against A/Victoria-infected cells, in contrast to normal subjects. SLE patients responded with levels of serum hemagglutination-inhibition antibody which were similar to those of normal subjects. Thus, SLE patients manifest decreased cell-mediated cytotoxicity against virus-infected target cells, although humoral antibody responses appeared to be intact. Studies of SLE patients with influenza may help to define the role of cell-mediated immunity in the pathogenesis of certain viral infections.     

Antibody-Dependent Cell-Mediated Cytotoxicity in Cattle: Activity Against 51Cr-Labeled Chicken Erythrocytes Coated with Protozoal Antigens

Bovine mononuclear cells in the presence of bovine anti-chicken erythrocyte sera at high dilutions induce release of chromium-51 from labeled chicken erythrocytes. Bovine effector cells are capable of recognizing both bovine immunoglobulin G(1) and bovine immunoglobulin G(2); in contrast, human effector cells only recognize immunoglobulin G(1). Effector cell activity of bovine mononuclear cells is equally distributed between peripheral blood and spleen. As in other species, thymus and lymph node cells exert no antibody-dependent effect, although some direct cytotoxicity by lymph node cells may be observed. Antibody-dependent cell-mediated cytotoxicity against a bovine cell line can also be detected. By using a tannic acid technique, it was found that chicken erythrocytes coated with Theileria parva piroplasm antigen or with Trypanosoma rhodesiense variant-specific coat antigen form suitable targets for bovine antibody-dependent cell-mediated cytotoxicity assays. By using such targets, a moderate degree of direct cytotoxicity by bovine mononuclear cells, in the absence of antibody, is always observed; this may be reduced by choosing optimal conditions of tannic acid treatment and antigen sensitization and by the use of short incubation periods for the cytotoxicity assay. Observations have been made on the variant specificity, time course of appearance, and association with immunoglobulin G(1) of the antibody activity responsible for cell-dependent cytotoxicity against chicken erythrocytes coated with T. rhodesiense antigens. The potential usefulness of this technique in the analysis of protective immune responses against protozoal infections is discussed.     

Antibody-dependent cell-mediated cytotoxicity: heterogeneity of effector cells in human peripheral blood.

We have compared antibody-dependent cell-mediated cytotoxicity (ADCMC) of human peripheral blood leukocytes (PBL) in three model systems. target cells were 51Cr-labeled mouse mastocytoma cells, chicken erythrocytes (CRBC), and human erythrocytes (HRBC) coated with appropriate heterologous or isologous antisera. Effector cells were characterized on the basis of their adherence, phagocytosis, radiosensitivity, and sedimentation velocity(s) at 1 g. In predominantly mononuclear (Ficoll-Isopaque-purified) PBL preparations (MPBL) HRBC were lysed by an adherent, phagocytic population of cells that was markedly radio-resistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions (s greater than 4.5 mm/hr). On the other hand, mastocytoma cells were lysed by a population of MPBL that was nonadherent, nonphagocytic, and relatively radiosensitive. These cells mainly restricted to slowly sedimenting fractions (s greater than 4.5 mm/hr) following 1 g velocity sedimentation. CRBC appeared to be susceptible to lysis by both types of mononuclear effector cell. In some experiments, enriched populations of polymorphonuclear leukocytes (PMN) were isolated. These cells were found to lyse both HRBC and CRBC very efficiently, whereas mastocytoma cells were lysed very little if at all by the same effector populations. Taken together, these results suggest that antibody-coated mastocytoma cells are lysed uniquely by effector cells in human peripheral blood with the physical properties of lymphocytes, whereas antibody-coated HRBC are lysed by both monocytes and PMN, but not by lymphocytes. Antibody-coated CRBC would appear to be lysed by all of the three effector cell types tested.     

Antibody-Dependent Cell-Mediated Cytotoxicity: Heterogeneity of Effector Cells in Human Peripheral Blood

We have compared antibody-dependent cell-mediated cytotoxicity (ADCMC) of human peripheral blood leukocytes (PBL) in three model systems Target cells were ⁵¹Cr-labeled mouse mastocytoma cells, chicken erythrocytes (CRBC), and human erythrocytes (HRBC) coated with appropriate heterologous or isologous antisera. Effector cells were characterized on the basis of their adherence, phagocytosis, radiosensitivity, and sedimentation velocity(s) at 1 g. In predominantly mononuclear (Ficoll-Isopaque-purified) PBL preparations (MPBL) HRBC were lysed by an adherent, phagocytic population of cells that was markedly radioresistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions ( s > 4.5 mm/hr). On the other hand, mastocytoma cells were lysed by a population of MPBL that was nonadherent, nonphagocytic, and relatively radiosensitive. These cells were mainly restricted to slowly sedimenting fractions ( s < 4.5 mm/hr) following 1 g. velocity sedimentation. CRBC appeared to be susceptible to lysis by both types of mononuclear effector cell. In some experiments, enriched populations of polymorphonuclear leukocytes (PMN) were isolated. These cells were found to lyse both HRBC and CRBC very efficiently, whereas mastocytoma cells were lysed very little if at all by the same effector populations. Taken together, these results suggest that antibody-coated mastocytoma cells are lysed uniquely by effector cells in human peripheral blood with the physical properties of lymphocytes, whereas antibody-coated HRBC are lysed by both monocytes and PMN, but not by lymphocytes. Antibody-coated CRBC would appear to be lysed by all of the three effector cell types tested.     

Human T-Lymphocyte Subpopulations: Alterations in Systemic Lupus Erythematosus

Sharply reduced proportions of T cells with Fc receptors for IgG (TG cells) were observed in blood samples from patients with systemic lupus erythematosus (SLE), mainly with active disease. This T-cell subset has previously been shown to be a suppressor in the pokeweed mitogen (PWM)-dependent B-cell differentiation. In contrast, the percentages of T cells with Fc receptors for IgM (TM cells), which have been shown to help immunoglobulin production, were not different from those of normals. TG cells present in the circulation of SLE patients were analysed for their functional capacities in antibody-dependent cell-mediated cytotoxicity and in the suppression of a PWM-induced B-cell differentiation. In both these assays TG cells from SLE patients had normal effector cell activity. This suggests that thr than a qualitative type.     

Characteristics of complexes for arming and inhibiting effector cells for antibody-dependent cell-mediated cytotoxicity.

The size of IgG aggregate effective in the inhibition or arming of human effector cells for antibody-dependent cell-mediated cytotoxicity (ADCC) was investigated using heat- and alkalipolymerized rabbit IgG or purified antibody fractionated by gel filtration. In contrast to the inhibition of ADCC against chicken erythrocytes, which was marked when effector cell were pre-incubated with high molecular weight aggregates (19S or greater), small polymers were most effective in arming for cytotoxicity against antigen-coated chicken red cells. Our data also demonstrate that while the cytotoxic potential of armed cells is short-lived and rapidly lost during culture at 37 degrees C but not 4 degrees C, the reduced capacity of these cells to kill antibody-coated targets is not altered by similar incubation at 37 degrees C. The differences in the size of aggregate active in arming and inhibition, and the stability of the two phenomena are compatible with the hypothesis that large aggregates may cause more cross-linking and redistribution of effector cell Fc receptors than small polymers of IgG.     

Autoantibodies against neutrophil cytoplasm components in systemic lupus erythematosus and in hydralazine-induced lupus.

Anti-neutrophil cytoplasm antibody (ANCA) has been shown to be no marker of systemic lupus erythematosus (SLE) including lupus nephritis or of progressive systemic sclerosis (PSS). Antibodies against myeloperoxidase (anti-MPO) and elastase, two granulocyte lysosomal enzymes, were found in patients with SLE but not in those with PSS, except for one patient who had anti-MPO. Anti-MPO was present in 21% of patients with SLE, and at low concentrations in about 80% of these cases. Anti-elastase was found in four patients with SLE. In another group of six patients with a SLE-like syndrome induced by anti-hypertensive treatment with the anti-hypertensive hydralazine, anti-MPO antibodies occurred in all six, and anti-elastase antibodies in five. Monitored during a 2-year follow-up period, anti-MPO antibodies were found to persist, whereas anti-elastase antibodies were rapidly eliminated, after withdrawal of the drug.     

Increased multiclonal antibody-forming cell activity in the peripheral blood of patients with SLE

21 patients with criteria for systemic lupus erythematosus (SLE) and 12 normal controls were studied for their spontaneous circulating IgM and IgG plaque-forming cells (PFCs) reactive against sheep erythrocytes (SRBC) and against a panel of five haptens. Quantitatively defined active and mildly active SLE patients had significantly elevated IgM- and IgG-producing PFCs in their peripheral blood reactive with the panel of five chemically defined haptens. Those patients having inactive SLE also showed increased circulating IgM PFCs. Significant elevations in circulating hapten-reactive PFCs were found to correlate progressively with disease activity in the inactive, mildly active, and active SLE patient groups. Circulating IgM- and IgG-secreting PFC reactive against SRBC were both significantly elevated only in those patients with active SLE. The data support the concept that SLE patients have a generalized increase in B cell activity against a broad repertoire of determinants, even those ostensibly unrelated to natural tissue antigens.     

Cytotoxicity of immune guinea-pig cells: I. Investigation of a correlation with delayed hypersensitivity and a comparison of cytotoxicity of spleen, lymph node and peritoneal exudate cells

The work was intended to show whether any correlation could be established between delayed hypersensitivity and the appearance of specifically cytotoxic cells in the spleen, lymph nodes and peritoneal exudate.

Using chicken erythrocytes as target cells, the cytotoxicity of cells from immunized guinea-pigs was assessed by the ⁵¹Cr release technique. After immunization with chicken erythrocytes and complete adjuvant, spleen, lymph node and peritoneal exudate cells were active, the overall time course of cytotoxicity corresponding broadly with intensity of delayed hypersensitivity reactions. Following intravenous immunization with erythrocytes, there was no delayed hypersensitivity response, and lymph node cells were not cytotoxic; spleen cells and peritoneal exudate cells showed cytotoxicity. A further partial correlation with delayed hypersensitivity was shown in immune-deviated guinea-pigs. Lymph node cell cytotoxicity and delayed hypersensitivity were markedly depressed while spleen cytotoxicity was unimpaired and peritoneal exudate cytotoxicity only slightly lowered.

Assessed by effector: target cell ratio, peritoneal exudate cells were found to produce greater cytotoxicity than spleen or lymph node cells. Partial cell separation by adherence showed that cytotoxicity was associated with peritoneal macrophages. Cytotoxicity directed against chicken erythrocytes was found to be target-cell specific.

     

Mechanisms of specific and non-specific tumour immunity after azathioprine treatment of mice.

The ability of phagocyte-depleted spleen cells to lyse chicken erythrocytes (CRBC) in the presence of antibody was measured in mice which had been treated with the antimetabolite azathioprine. Single doses of the drug had no effect on this ability when measured on the day after administration. A 4-day course of 80 mg/kg/day of the drug markedly reduced splenic antibody-dependent cell-mediated cytotoxicity (ADCMC) although it reduced neither antibody responses nor the development of cytotoxic cells following subsequent immunization with an allogeneic tumour. Splenic phagocytosis and phagocyte-mediated ADCMC were both slightly enhanced following drug treatment. The implications of these findings are that the major antibody-dependent cytotoxic cell in phagocyte-depleted mouse spleen is normally in a state of proliferation, and plays no important role in antigen recognition.     

Heterogeneous neurocytotoxic antibodies in systemic lupus erythematosus.

Sera from eighteen patients with systemic lupus erythematosus (SLE) were tested for cytotoxic antibody to three neuronal and two glial continuous cell lines of human origin. Eighty per cent (fifteen out of eighteen) of the sera were cytotoxic to at least one of the cell lines, but only seven sera were active against all five lines. Three sera had anti-neuronal but not anti-glial reactivity. No sera were gliocytotoxic without neurocytotoxicity. Three SLE sera with relatively strong cytotoxicity to all five cell lines were abosrbed with each of the cell lines separately and the absorbed sera were then tested for residual cytotoxicity to each of the cell lines. The absorptions uncovered at least six different antibody specificities directed at antigens expressed on some but not all of the neuronal and glial cell lines. Each patient's serum had its own profile of antibody specificities reactive with membrane antigens on nervous tissue-derived cells.     

Autoantibodies against eukaryotic protein L7 in patients suffering from systemic lupus erythematosus and progressive systemic sclerosis: frequency and correlation with clinical, serological and genetic parameters. The SLE Study Group.

Recent studies have shown that sera of patients suffering from systemic autoimmune diseases contain autoantibodies directed against the eukaryotic ribosomal protein L7 [1]. In the present study we screened a large panel of sera from patients with systemic lupus erythematosus (SLE) for the presence of anti-L7 autoantibodies and their relationship to clinical, serological and genetic parameters of SLE. By means of an ELISA employing recombinant protein L7 as antigen we detected anti-L7 autoantobodies in 172 of 506 SLE sera (34%). Negative correlations were observed between the presence of anti-L7 autoantibodies, serum IgG levels and proteinuria; a potentially positive relationship existed with lung fibrosis. In order to analyse further this possibility we screened sera of 129 patients suffering from progressive systemic sclerosis (PSS) for anti-L7 reactivity; 45 of these patients had lung fibrosis. Of the PSS patients, 41% exhibited anti-L7 autoantibodies, but positive reactions were evenly distributed among patients with and without lung fibrosis. Protein L7 thus represents a major autoantigen of systemic autoimmune diseases, but does not so far define a distinct subpopulation of patients.     

Natural cytotoxicity in systemic lupus erythematosus: mechanisms of suppression by inhibitory serum factors.

Spontaneous cytotoxicity mediated by natural killer (NK) cells is impaired in several human diseases including systemic lupus erythematosus (SLE). The present study was designed to describe factors in SLE sera which suppress the NK function of unfractionated mononuclear cells and NK enriched suspensions. NK activity was determined in 19 SLE patients and 25 normal controls by a standard chromium release assay. Sera obtained from SLE patients suppressed normal NK activity by an average of 29.4%. The presence of anti-lymphocyte antibodies (ALA) of the IgM class which were reactive with unfractionated mononuclear cells or the NK cell enriched OKM1 positive subset correlated with serum-mediated suppression. NK inhibitory SLE sera did not interfere with normal effector-target conjugate formation. These results demonstrate the modulatory effects of immune aggregates and ALA on lymphocyte function in SLE. These factors suppress NK function without evidence of lymphocyte cell death or inhibition of NK effector cell binding to tumour targets.