Antibody-dependent cellular cytotoxicity of vascular endothelium: characterization and pathogenic associations in systemic sclerosis.

Ten sera from 48 patients with systemic sclerosis were found to be capable of producing cytotoxicity of human umbilical venous and arterial endothelium when co-cultured with peripheral blood mononuclear cells. Fractionation of sera on Ultrogel and the preparation of monomeric IgG by ion exchange and affinity chromatography suggested that the cytotoxicity was mediated by anti-endothelial antibodies capable of pre-sensitizing target cells in a mechanism that resembled antibody-dependent cellular cytotoxicity. These anti-endothelial antibodies together with C1q-binding immune complexes and anti-cardiolipin antibodies were found in 18 of 28 patients so investigated, suggesting that multiple immunological mechanisms may be involved in the pathogenesis of the vascular lesion of systemic sclerosis.     

Antibody-dependent cellular cytotoxicity in asthmatics.

Using a 51Cr-labeled, antibody-coated chicken red blood cell assay system, mononuclear cell and granulocyte preparations from infectious asthmatics, noinfectious asthmatics, and normal controls were tested for antibody-dependent cellular cytotoxicity (ADCC) capacity. The corrected cytotoxic indices of mononuclear cell preparations from infectious asthmatics were reduced (34 +/- 10 SD) as compared to noninfectious asthmatiics (47 +/- 7 SD) or normal controls (47 +/- 6 SD). Granulocyte preparations from infectious asthmatics and normal controls had a severity of the disease or in vivo drug treatment.     

Antibody-dependent cellular cytotoxicity in poikilotherms

⁵¹Cr-chromate labelled chicken red blood cells, treated with rabbit (anti-chicken red blood cell) serum, are lysed in vitro, in the absence of complement, by spleen cells from Xenopus laevis, Ambystoma mexicanum or Lacerta viridis. Optimal conditions for lysis by Xenopus spleen cells were determined. The phenomenon seems homologous with antibody-dependent cellular cytotoxicity (ADCC) mediated by mammalian or avian K cells. The phylogenetic significance of the finding is discussed.     

Selective decrease in antibody-dependent cell-mediated cytotoxicity in systemic lupus erythematosus and progressive systemic sclerosis.

With the use of two target cells (chicken erythrocytes and Chang cells), the antibody-dependent cell-mediated cytotoxicity (ADCMC) of peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS) was studied. Patients with active SLE had a significant reduction in ADCMC against Chang cells whereas cytotoxicity against chicken erythrocytes did not differ significantly from that of a control population. Similarly, a group of PSS patients with positive anti-DNP antibodies demonstrated a selective reduction in ADCMC against Chang cells. These findings support the concept that different effector cells mediate ADCMC against chicken erythrocytes and Chang cells, and indicate that in some patients with SLE and PSS there is a selective reduction or blockade of the ADCMC effector cell active against Chang cells.     

Antibody-dependent cellular cytotoxicity in recurrent aphthous ulceration.

Antibody-dependent cellular cytotoxicity (ADCC) was investigated as s possible mechanism of pathogenesis in recurrent aphthous ulceration (RAU). ADCC induced by mononuclear cells from patients at various stages of RAU was compared with ADCC induced by mononuclear cells from paired controls. Chicken red blood cells (ChRBC) coated with rabbit anti-ChRBC antibody were used as targets. A significant increase in ADCC (P less than 0.01, paired sample t-test) in patients' samples as compared with controls was found only at the early stage of the disease. No increased cytotoxicity over control values was observed at any other single stage of the disease. The transient increased cytotoxicity observed in the early stage of RAU may reflect a number of possible mechanisms, including an increased number of ADCC effector cells only at the early stage, an increase in Fc receptor avidity, or an increase in efficiency of the existing effector cell population.     

Antibody-dependent cell-mediated cytotoxicity to Varicella zoster.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against Varicella zoster (VZ) infected fibroblasts is described. ADCC requires antibody to VZ and is greater with heavily infected targets. It is not dependent on the immune status of the effector cells. The effector cells responsible for ADCC are present in sheep red cell (E) positive and E-fractions of peripheral blood, and in the G10 non-adherent population. The ADCC activity is present in microexudate non-adherent cells and in the adherent population to a lesser extent. The technique provides a means to study host defence against VZ infection in immune compromised individuals.     

Altered B lymphocyte function induces systemic autoimmunity in systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs. SSc is associated with immune activation characterized by autoantibody production, lymphocyte activation, and release of various cytokines. The presence of autoantibodies is a central feature of immune activation in SSc. Although autoantibodies are thought to be closely linked to the pathogenesis of SSc, the pathogenic relationship between systemic autoimmunity and the clinical manifestations of SSc, including skin fibrosis, remains unknown. Recent studies have revealed that B cells play a critical role in systemic autoimmunity and disease expression through various functions, including cytokine production in addition to autoantibody production. The B cell signaling thresholds are regulated by response regulators that augment or diminish B cell signals during responses to self and foreign antigens. Abnormal regulation of the response regulator function and expression may result in autoantibody production. Among these response regulators, CD19, which is a critical cell-surface signal transduction molecule of B cells, is the most potent positive regulator. Transgenic mice that overexpress CD19 by 3-fold lose tolerance and generate autoantibodies spontaneously. B cells from SSc patients exhibit a 20%-increase in CD19 expression that induces SSc-specific autoantibody production in transgenic mice. Furthermore, SSc patients have intrinsic B cell abnormalities characterized by expanded naive B cells, activated but diminished memory B cells, and chronic hyper-reactivity of memory B cells, possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a model of SSc, show augmented CD19 signaling and chronic hyper-reactivity. Remarkably, CD19 loss results in inhibition of chronic B cell hyper-reactivity and elimination of autoantibody production, which is associated with improvement in skin fibrosis and a parallel decrease in IL-6 production by B cells. Thus, chronic B cell activation resulting from augmented CD19 signaling leads to skin fibrosis possibly through IL-6 overproduction, as well as autoantibody production, in tight-skin mice and SSc patients.     

Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis.

Enhanced natural killer (NK) activity and normal lymphocyte antibody-dependent cellular cytotoxicity (ADCC) were observed in 16 patients with a diagnosis of progressive systemic sclerosis (PSS). Higher NK activity levels were observed against NK-sensitive K562 target cells, while the NK-resistant P815, Daudi and Raji cell lines were not lysed. Cytofluorimetric studies and morphological analysis of peripheral blood lymphocytes (PBL) showed an increased number of CD16 positive cells and large granular lymphocytes (LGL), indicating that the enhancement observed was probably attributable to an increase in the number of circulating NK cells.     

Altered B lymphocyte homeostasis and functions in systemic sclerosis

Beyond the production of autoantibodies, B-cells are thought to play a role in systemic sclerosis (SSc) by secreting proinflammatory/profibrotic cytokines. B-cells are a heterogeneous population with different subsets distinguished by their phenotypes and cytokine production. Data about B-cell subsets, cytokine production and intracellular pathways leading to this production are scarce in SSc. The aim of our study was to describe B-cell homeostasis, activation, proliferation, cytokine production in B-cells and serum and B-cell intracellular signaling pathways in SSc. We hypothezided that B-cell homeostasis and cytokine production were altered in SSc and could be explained by serum cytokine as well as by intracellular signaling pathway abnormalities.Forty SSc patients and 20 healthy controls (HC) were prospectively included. B-cell subsets were determined by flow cytometry using CD19, CD21, CD24, CD38, CD27, IgM and IgD. CD25, CD80, CD95, HLA-DR were used to assess B-cell activation. Intracellular production of IL-10 and IL-6 were assessed by flow cytometry after TLR9 and CD40 stimulation. IL-6, IL-10, Ki67, Bcl2 mRNA were quantified in B-cells. Cytokine production was also assessed in sera and supernatants of B-cell culture, using a multiplex approach. Signaling pathways were studied through phosphorylation of mTOR, ERK, STAT3, STAT5 using a flow cytometry approach.We found that SSc patients exhibited an altered peripheral blood B-cell subset distribution, with decreased memory B-cells but increased proportion of naive and CD21^LoCD38^Lo B-cell subsets. We observed an increased expression of activation markers (CD80, CD95, HLA-DR) on some B-cell subsets, mainly the memory B-cells. Secretion of IL-6, BAFF and CXCL13 were increased in SSc sera. There was no correlation between the peripheral blood B-cell subsets and the serum concentrations of these cytokines. After stimulation, we observed a lower proportion of IL-10 and IL-6 producing B–cells in SSc. Finally, we observed a significant decrease of mTOR phosphorylation in SSc patient B-cells.In conclusion, we observed an altered B-cell homeostasis in SSc patients compared to HC. Memory B-cells were both decreased and activated in patients. IL-10 producing B-cells were decreased in SSc. This decrease was associated with an alteration of mTOR phosphorylation in B-cells. Conversely, there was no correlation between serum cytokine profile and B-cell homeostasis alterations.     

T lymphocyte abnormalities in juvenile systemic sclerosis patients

Multi-center evaluations of pediatric patients with juvenile systemic sclerosis (jSSc) have suggested that the pathogenesis of jSSc may differ from that of systemic sclerosis (SSc) in adult patients. Therefore, we undertook to identify abnormalities in the T lymphocytes of jSSc patients and to determine if they differed from the abnormalities reported in the T lymphocytes of adult SSc patients. We identified decreases in the frequency of resting regulatory T lymphocytes and an increased frequency of CD45RA expressing effector memory (EMRA) CD4 T lymphocytes, which were characterized by an increased frequency of CCR7 protein expressing cells. Neither the increases in the EMRA subpopulation nor the increased CCR7 protein expression have been reported in adult SSc patients. The decrease in resting regulatory T lymphocytes in jSSc patients may permit the expansion of the disease initiating CD4 T lymphocytes present in the CCR7 expressing EMRA CD4 T lymphocyte subpopulation.     

Antibody-dependent cell-mediated cytotoxicity in Graves disease

The antibody-dependent cell-mediated cytotoxicity of peripheral lymphocytes from patients with Graves disease was tested using chicken erythrocytes coated with rabbit antichicken red blood cell antibody and labelled with 51Cr. An increased cytotoxic activity was found in untreated Graves disease without ophthalmopathy. The cytotoxic activity was less elevated in the groups of patients with severe ophthalmopathy or high level of anti-thyroglobulin antibody, and approximately normal after methimazole treatment.     

Antibody-dependent cell-mediated cytotoxicity against varicella-zoster virus-infected targets.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against cryopreserved varicella-zoster virus-infected human foreskin fibroblasts was detected in a 51Cr release assay. Target cells, samples of seropositive or seronegative sera, and mononuclear cells obtained by Ficoll-Hypaque centrifugation of human peripheral blood were added to microtiter plate wells and allowed to incubate at 37 degrees C for 4 h. Fibroblasts infected for 48 to 96 h were susceptible to ADCC. Effector cells from seropositive and seronegative normal children were equally active in the assay. Antibody titers were determined by testing serial dilutions of sera in the ADCC assay. Zoster immune globulin had a titer of 204,800. Sera from 40 naturally seropositive individuals were compared by assays for ADCC and fluorescent antibody to membrane antigen. All sera that were negative by fluorescent antibody to membrane antigen (less than 2) were also negative by ADCC (less than 20). All sera that were positive by fluorescent antibody to membrane antigen were also positive by ADCC, but titers of individual sera were frequently 5 to 20 times higher in the ADCC assay.     

Antibody-dependent lymphocyte killer function in human immunodeficiency diseases.

Antibody-dependent cell immunity to the lymphocyte system (ABCIL) has been shown to be a function of a non-thymus-processed cell in the experimental animal. To evaluate its role in the human and to assess its clinical usefulness, we assessed ABCIL in twenty-five patients with various immunodeficiency (ID) syndromes. Our technique measures the lysis of 51Cr-labelled normal human lymphocytes coated with HL-A-specific antibody. Cytotoxicity is expressed as a percentage of 51Cr released after subtracting the spontaneous target cell release. Mean values in normals are 20+/-2 (s.e.). The ten patients with AB deficiency had a mean ABCIL of 7-9+/-2 (Pless than0-01). All eight patients with cellular ID had normal ABCIL (18+/-2), while the ten patients with combined ID had variable results. Effector cell function in the ABCIL test correlated (r=0-74; Pless than0-05) with the percentage of B cells in the peripheral blood. No correlation was found between ABCIL function and serum immunoglobulin levels or rosette-forming cells in the peripheral blood. There is a function for B lymphocytes other than as a precursor of antibody-synthesizing cells.     

An absolute requirement for serum macromolecules in phytohaemagglutinin-induced human lymphocyte DNA synthesis.

We have examined the effect of different variables such as tissue culture media, with or without various supplements, lymphocyte isolation techniques, lymphocyte contamination by autologous red blood cells and platelets, and lymphocyte numbers, on the requirement for serum during phytohaemagglutinin (PHA) induced DNA synthesis in human lymphocytes. At all mitogen doses tested, we have found that dialysable constituents of serum enrich the ability of all tissue culture media to support lymphocyte DNA synthesis; however, human lymphocytes display an absolute requirement for nondialysable macromolecular constituents of serum in order to synthesize DNA.     

Antibody-dependent macrophage-mediated cytotoxicity against Entamoeba histolytica

Interactions between trophozoites of Entamoeba histolytica and peritoneal exudate macrophages from unsensitised and antigen-sensitised animals were studied in vitro. Normal macrophages killed trophozoites to some extent. This killing capacity was enhanced by prior sensitisation of the animals with specific antigen. Incorporation of anti-amoebic antiserum in the amoeba-macrophage mixture greatly enhanced the killing capacity of macrophages. Fraction one (F-I) of a crude amoebic extract was most effective in enhancing the cytotoxicity of macrophages by prior sensitisation and anti-F-I serum was the most effective antiserum. The cytotoxicity-inducing capacity of the immune serum resided in the IgG but not in the IgM fraction.     

Antibody-dependent cell-mediated cytotoxicity in the rat. The role of macrophages.

Experiments are described in which the inactivation of macrophage activity by quartz particles is used to investigate the role of macrophages in antibody-dependent cell-mediated cytotoxicity in the rat. The results confirm previous findings which indicated that macrophages play no significant role in assays using cell line cells as targets. On the other hand, previous suggestions that macrophages are active against antibody-coated chick erythrocytes cannot be substantiated. In fact, it is shown that macrophages can play a protective role, and that inhibiting macrophage activity in peritoneal exudates leads to a spectacular increase in antibody-dependent lysis of chick erythrocytes. It is suggested that the confusion surrounding the role of macrophages in this assay has resulted from the failure to recognize that adherence techniques such as carbonyl-iron treatment of cell suspensions can result in substantial depletion of non-phagocytic adherent cells.     

Antibody-dependent cell-mediated cytotoxicity againstEscherichia coli O antigens

Antibodies againstEscherichia coli O antigen from rabbits immunized with formalin-killed bacteria were tested for cytotoxic capacity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay with human lymphocytes as effector cells and autologous papainized erythrocytes coated with O antigen as target cells. The cytotoxic titres were compared with the titres obtained with three methods of antibody quantitation. It was found that ADCC recorded antibodies with similar sensitivity as the enzyme-linked immunosorbent assay (ELISA) for IgG, but was much more sensitive than the ammonium sulphate precipitation (ASP) and indirect haemagglutination (IHA) usingβ-mercaptoethanol reduced sera. The ADCC titres were found to correlate very well with the titres obtained with ASP, ELISA and IHA for IgG but not for IgM, which is in accordance with a previous notion that ADCC is primarily mediated via IgG antibodies. ADCC should be considered as a possible immunopathologic mechanism in renal parenchymal damage in connection with urinary tract infections.     

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.