calcimycin and Lupus-Erythematosus--Systemic

Numerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-kappa B contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-kappa B protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-kappa B binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-kappa B activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-kappa B binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-kappa B activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-kappa B activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

Topics: Autoradiography; Calcimycin; DNA-Binding Proteins; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Lupus Erythematosus, Systemic; Lymphocyte Activation; NF-kappa B; NF-kappa B p50 Subunit; Normal Distribution; Receptors, Antigen, T-Cell; Severity of Illness Index; T-Lymphocytes; Tetradecanoylphorbol Acetate; Time Factors; Transcription Factor RelA

Two murine models of lupus were employed to challenge an hypothesized mechanism by which antiphospholipid antibodies (APLA) might promote thrombosis: altering prostacyclin (PGI2) and thromboxane (TX) production. PGI2 levels in mouse blood and the ex vivo release of PGI2 and TX from mouse kidney were measured. Since APLA have been reported to alter synthesis or activation of several molecules mediating fibrinolysis, murine plasma levels of the fibrin degradation product, D-dimer were also determined. Two murine strains, one prone to spontaneous "lupus-like" illness (MRL-lpr) and related strain (MRL-(+2)), were compared. The assays confirm that MRL-lpr mice have increased anticardiolipin antibody (ACA) and two-fold increased release of TX from renal tissues compared to MRL-(+2) mice. However, these mice have low levels of plasma D-dimer. NIH Swiss mice injected with IgG (containing APLA) from thrombosis-prone lupus patients had high blood ACA titers and D-dimer levels, but both ACA and D-dimer were low or non-detectable in Swiss mice injected with saline or normal IgG. Unlike mice with spontaneous lupus-like illness, healthy mice injected with APLA did not differ from controls with respect to plasma or tissue PGI2 or TX levels. The two murine models of lupus differ, because an altered PGI2-TX ratio is a finding in the chronic murine lupus strain MRL-lpr, but is not seen when APLA are injected into normal mice. It is unlikely that APLA alone has a direct effect on cellular production of eicosanoids in vivo.

Topics: Animals; Antibodies, Antiphospholipid; Antiphospholipid Syndrome; Calcimycin; Disease Models, Animal; Eicosanoids; Epoprostenol; Female; Fibrin Fibrinogen Degradation Products; Humans; Immunoglobulin G; Kidney; Lupus Erythematosus, Systemic; Male; Mice; Mice, Inbred Strains; Thrombin; Thromboxane B2

Several immuno-regulatory abnormalities have been described in SLE patients. T cell dysfunction in SLE includes defective in vitro proliferative responses to several stimuli, reduced IL-2 production and a poor helper function. It has been widely proposed that this defective T cell immunoregulatory function has a key role in the hyperactivity of B cells and auto-antibody production in SLE. However, it has not been elucidated whether or not this cell dysfunction is intrinsic to lymphocytes or is due to other factors such as anti-lymphocyte auto-antibodies. In this study we have evaluated some important early cell activation events in T and non-T lymphocytes from patients with systemic lupus erythematosus (SLE). Peripheral blood lymphocytes from SLE patients and controls were isolated. The intracellular pH (pHi), cytosolic calcium (Ca2+i) and CD69 expression were determined by spectrofluorometry and flow cytometry. Modifications of these parameters in response to protein kinase C (PKC) activators, mitogenic lectins and calcium ionophores were also studied. We found a significant reduction in the increase of pHi in response to PKC activators (PMA) in SLE cells. In addition, the induction of CD69 expression by PMA was significantly lower in T cells from SLE patients. By contrast, freshly isolated non-stimulated SLE cells exhibited a significantly higher pHi, as well as an increased baseline expression of the early cell activation antigen CD69. On the other hand, the increase in Ca2+i in response to a Ca2+ ionophore (4Br-A23187) or thapsigargin in Ca(2+)-free solutions, was smaller in SLE lymphocytes. We concluded that T cells from SLE patients exhibit abnormalities in several key early cell activation events (pHi, Ca2+i and CD69 expression). These abnormalities could have an important role in the T cell dysfunction observed in SLE. The presence of T cells with a preactivated phenotype in the peripheral blood of SLE patients, could be a reflection of the ongoing autoimmune phenomena that is occurring in these patients.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Calcimycin; Calcium; Carcinogens; Cell Division; Cytosol; Enzyme Inhibitors; Female; Flow Cytometry; Humans; Hydrogen-Ion Concentration; Interleukin-2; Intracellular Fluid; Ionophores; Lectins, C-Type; Lupus Erythematosus, Systemic; Lymphocyte Activation; Protein Kinase C; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thapsigargin

Anti-platelet autoantibodies may be responsible for hematological complications in patients with systemic lupus erythematosus (SLE), but the mechanisms by which these antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma autoantibody, 9604, recognizes a surface antigen expressed on platelets activated by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited by EDTA, indicating partial dependence on divalent cations. It did not appear to be dependent on platelet secretion, nor did it directly affect alpha-granule or dense granule secretion. The protein antigen responsible for the binding of 9604 to activated platelets was characterized by Western blot and immunoprecipitation and shown to have a native molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32). Antibody 9604 had little or no effect on the shape change and the initial rate of primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation of stirred platelet suspensions (IC50 < or = 1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation (secondary aggregation), with a major shift to smaller microaggregates and singlets, was confirmed by direct particle count and sizing studies. The functional inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests one potential mechanism that may play a role in the hemostatic disorders found in SLE.

Topics: Adenosine Diphosphate; Antigens, Surface; Autoantibodies; Calcimycin; Flow Cytometry; Humans; Hybridomas; Lupus Erythematosus, Systemic; Phorbol Esters; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors

In our previous report, we demonstrated that the functions of phagocytes and lymphocytes were defective in patients with systemic lupus erythematosus (SLE). In an attempt to further clarify the defective mechanisms of these cells, 25 active SLE, 10 bronchial asthma patients (BA) on corticosteroids and 25 age and sex-matched normal individuals were investigated for the expression of membraneous C3b receptors, ionophore-induced 45Ca(2+)-uptake, mitochondrial potentials and phagocytic activity of neutrophils. We found decreased expression of C3b receptors on SLE PMN in both resting (37.2 +/- 3.7% of the normal controls) and FMLP-stimulated (68.3 +/- 7.1% of the normal controls) conditions, whereas the C3b receptor expression on BA-PMN receiving long-term steroid treatment was not different from normal controls. This suggests that the defective phagocytosis of SLE PMN is in the recognition, but not in the ingestion phase because of the normal function of Ca(2+)-influx and mitochondrial activity in SLE PMN. On the other hand, hyporesponsiveness to PHA stimulation (stimulation index: 127.4 +/- 46.3 in SLE vs. 311.2 +/- 30.4 in normals, p = 0.0077) was a distinct cell-mediated immune abnormality in our SLE patients. We measured the membrane potential of individual cells using 3,3'-dihexyloxacarbocyanin and found hyperpolarization in resting SLE lymphocytes. However, the membrane polarization of SLE lymphocytes became lower than that of normal cells after PHA stimulation for 3 days. A similar tendency was also found in Na(+)-K(+)-dependent ATPase activity in SLE lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adolescent; Adult; Asthma; Calcimycin; Calcium; Disease Susceptibility; Female; Humans; Immunologic Deficiency Syndromes; Infections; Lupus Erythematosus, Systemic; Lymphocyte Activation; Lymphocytes; Membrane Potentials; Middle Aged; Neutrophils; Phagocytosis; Receptors, Complement; Receptors, Complement 3b; Sodium-Potassium-Exchanging ATPase

CD4+ cells from autoimmune-prone C57BL/6 lpr/lpr mice contain two subpopulations, B220-CD4+ and B220+CD4+ cells. Highly purified B220-CD4+ cells from C57BL/6 +/+ and lpr/lpr mice were examined by comparing functional characteristics and expression of cell surface antigens and T cell receptor (TcR)/CD3 complex. Both lpr B220+CD4+ and B220+CD4-CD8- cells, most of which were PgP-1 positive, expressed TcR/CD3 complex on the cell surface at lower level as compared with B220-CD4+ cells of age-matched normal mice. In addition, the B2200-CD4+ cells were heterogeneous on the basis of surface expression of PgP-1 and CD3 antigens. Normal levels of TcR C alpha-, C beta- and V beta 8-specific mRNA were found in the B220-CD4+ cells and B220+CD4+ cells as compared with normal B220-CD4+ cells, while V beta 8-specific mRNA was preferentially expressed only by B220+CD4-CD8- cells. Either B220+CD4+ cells and B220+CD4-CD8- cells failed to respond to anti-CD3 monoclonal antibody (MoAb) as assessed by proliferative responses and production of interleukin-2 (IL-2). However, appreciable levels of reactivity to anti-CD3 MoAb were detected in the B220-CD4+ cells, although the responsiveness of this subset to such stimuli were reduced, compared with those of normal control. These results indicate that the B220-CD4+ cells in lpr mice are phenotypically and functionally distinct from normal B220-CD4+ cells.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Blotting, Northern; Calcimycin; CD3 Complex; CD4 Antigens; CD4-Positive T-Lymphocytes; Flow Cytometry; Gene Expression; Interleukin-2; Leukocyte Common Antigens; Lupus Erythematosus, Systemic; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Mutant Strains; Receptors, Antigen, T-Cell; RNA, Messenger; Tetradecanoylphorbol Acetate

Interleukin-2 (IL-2) production was studied in T lymphocytes from 32 patients with systemic lupus erythematosus (SLE) and 27 healthy volunteers. The IL-2 production by phytohemagglutinin (PHA)-stimulated cells from SLE patients was significantly depressed compared to control values, with a correlation between degree of depression and disease activity. The depressed IL-2 production by SLE T cells are largely reversed by the addition of either phorbol ester (PMA) or partially by a calcium ionophore. SLE T cells had significantly lower peak increases in intracellular free calcium [( Ca2+]i) than controls after stimulation by PHA or by a monoclonal antibody against the CD3 antigen. This abnormality was found even in T cells from patients with mild disease activity or in those whose T cells produced normal amounts of IL-2. Calcium ionophore produced similar increases in [Ca2+]i in SLE patients as in normals. These results suggest that a major component of the defect responsible for decreased IL-2 production by SLE lymphocytes is proximal to protein kinase C activation and may involve impaired signal transduction after activation of the antigen receptor complex.

Topics: Adult; Calcimycin; Calcium; Cell Separation; Cells, Cultured; Female; Humans; Interleukin-2; Lupus Erythematosus, Systemic; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; T-Lymphocytes; Tetradecanoylphorbol Acetate

Peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS), Reiter's disease, osteoarthritis, and from healthy volunteers were investigated for interferon-gamma (IFN-gamma) production after mitogen activation. Phytohaemagglutinin stimulation revealed an impaired IFN-gamma production in RA, SLE, and PSS but normal levels in Reiter's disease and osteoarthritis. In RA this deficiency was also seen after pokeweed mitogen, OKT3, and concanavalin A activation. No major differences were found in interleukin 2 (IL-2) production and cell proliferation. The IL-2 receptor expression was reduced on stimulated RA lymphocytes. The deficient IFN-gamma production was compensated in RA by co-stimulation of PHA or OKT3 with phorbol myristic acetate (PMA). In addition, the combination of the calcium ionophore A 23187 and PMA induced a strong IFN-gamma secretion in all patient groups and in the controls.

Topics: Adolescent; Adult; Aged; Arthritis, Reactive; Arthritis, Rheumatoid; Autoimmune Diseases; Calcimycin; Drug Synergism; Humans; Interferon-gamma; Lectins; Lupus Erythematosus, Systemic; Lymphocyte Activation; Middle Aged; Osteoarthritis; Receptors, Immunologic; Receptors, Interleukin-2; Scleroderma, Systemic; Tetradecanoylphorbol Acetate

Topics: Adult; Calcimycin; Female; Humans; Lupus Erythematosus, Systemic; Male; Monocytes; Prednisolone; SRS-A

Mice homozygous for the lpr gene spontaneously develop massive lymphoproliferation and an associated lupus-like autoimmune disease. In addition, the total lymphoid organs from these mice express high levels of mRNA for the c-myb proto-oncogene. Since enhanced c-myb mRNA is normally observed in immature thymic lymphocytes but not normal peripheral T cells, this may be indicative of the abnormal maturation state of lpr T lymphocytes. To determine whether the abnormal Lyt-2-, L3T4- (double negative) T lymphocytes in lpr mice express high c-myb, we purified this population by complement-mediated lysis with anti-L3T4 and Lyt-2 antibody from B6/lpr lymph nodes. We found that increased c-myb mRNA is expressed by this double-negative subset. To assess whether the high level of c-myb correlated with the aberrant undifferentiated state of these cells, we examined the effects of T cell differentiation inducers, phorbol ester and calcium ionophore, on c-myb expression. We found that c-myb levels were depressed after phorbol ester and calcium ionophore treatment. Concomitantly, transcriptional activation of the interleukin 2 receptor gene and progression of these cells through the cell cycle were observed. Thus, in B6/lpr double-negative T cells, the regulation of c-myb, interleukin 2 receptor, and cell proliferation may be interrelated. A combination of Northern hybridization and nuclear run-on transcription assays revealed two levels at which c-myb can be regulated in the double-negative T cell subset. The gene is transcriptionally regulated in untreated cells, but on induction with phorbol ester and calcium ionophore, the gene is negatively regulated via post-transcriptional mechanisms.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Calcimycin; Gene Expression Regulation; Interleukin-2; Lupus Erythematosus, Systemic; Lymph Nodes; Lymphocyte Activation; Mice; Mice, Mutant Strains; Proto-Oncogene Proteins; Proto-Oncogenes; Receptors, Immunologic; Receptors, Interleukin-2; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Transcription, Genetic

A 37-year-old female who suffered from SLE had a bleeding disorder. At the time of initial evaluation, the main disease demonstrated was a delta-storage pool deficiency. After this improved, a marked decrease of aggregation still remained, when induced by either ADP, epinephrine, collagen, A23187, thrombin, or PAF-acether. Although arachidonate-induced aggregation was slightly decreased, thromboxane B2 was produced normally in response to exogenous arachidonate. The patient's endoperoxides and/or thromboxane A2 aggregated aspirin-treated platelets, though her platelets were themselves unresponsive. Impaired aggregability induced by TPA (12-0-tetradecanoylphorbol-13-acetate) or OAG (1-oleoyl-2-acetyl-glycerol) was also found. However, the phosphorylation of P43 and P20 induced by several stimulators including CA++ ionophore was normal, using 32P-labelled platelets. It is suggested that TPA or OAG-induced platelet aggregation requires not only the phosphorylation of those proteins, but also another unknown mechanism after the phosphorylation, and that the platelet dysfunction of this patient was due to a defect of some mechanism involving Ca++ uptake or mobilization of cytoplasmic Ca++ from intracellular storage sites.

Topics: Adenosine Diphosphate; Adult; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Blood Proteins; Calcimycin; Collagen; Diglycerides; Epinephrine; Female; Humans; Lupus Erythematosus, Systemic; Phorbols; Phosphorylation; Platelet Activating Factor; Platelet Aggregation; Tetradecanoylphorbol Acetate; Thrombin

The mechanism of poor lymphocyte transformation to mitogens was studied in selected patients with rheumatoid arthritis and systemic lupus erythematosus. Low lymphocyte response to PHA and Con-A in media containing autologous and homologous sera was usually associated with poor response to the calcium ionophore A23187, which induces blastogenesis by a different mechanism. The low lymphocyte response to mitogens in patients with rheumatoid arthritis could be restored by in vivo treatment with the anthelmintic drug, levamisole. The present findings suggest that intrinsic defects are responsible for the decreased cellular response in patients with rheumatoid arthritis and systemic lupus erythematosus.

Topics: Adolescent; Adult; Arthritis, Rheumatoid; Calcimycin; Concanavalin A; Humans; Lectins; Levamisole; Lupus Erythematosus, Systemic; Lymphocyte Activation; Middle Aged