The in vitro production and regulation of anti-double stranded DNA antibodies by peripheral blood mononuclear cells from normals and patients with systemic lupus erythematosus.

The in vitro production of anti-double stranded DNA antibodies (anti-DNA) by peripheral blood mononuclear cells (PBMC) was investigated in 19 patients with systemic lupus erythematosus (SLE) and in 12 normal individuals, using a micro solid phase enzyme immunoassay. PBMC from SLE patients spontaneously produced anti-DNA with a higher frequency (16 of 19) than did PBMC of controls (three of 12). In addition SLE patients produced predominantly IgG antibodies. PWM and DNA enhanced anti-DNA synthesis is spontaneously low and non-producers, but acted as inhibitors in spontaneously high producers. The partial removal of T cells decreased or abolished anti-DNA synthesis in four of nine SLE patients. In contrast the B cell enriched fractions of five of nine SLE and five of seven normal patients produced the same or higher anti-DNA levels than did the corresponding unseparated PBMC. These results suggest evidence for autoreactive B cells in SLE as well as in normals, and therefore the combination of these autoreactive B cells with helper and/or suppressor T cell disorders could lead to the over production of anti-DNA seen in different patients with SLE.     

The induction of human antinuclear antibodies by D-penicillamine: activation of inducer helper T cells in the absence of irradiation sensitive suppressor T cells.

The capacity of D-Penicillamine (DP) to induce or to potentiate the production of antinuclear antibodies (ANA), detected by immunofluorescence (IF), was investigated in vitro, using peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE) and normal individuals. Except in one patient with SLE, DP did not enhance ANA synthesis when using unseparated PBMC. In contrast, when B cells were cocultured with irradiated T cells or irradiated enriched T4+ subset, DP induced or potentiated the production of ANA. These results indicate that DP acts by stimulating T4+ helper cells to promote ANA synthesis in the absence of radio-sensitive suppressor T cell function contained within the T4+ population.     

Abnormal production of B cell growth factor in patients with systemic lupus erythematosus.

In order to clarify the role of B cell growth factor (BCGF) in the pathogenesis of systemic lupus erythematosus (SLE), BCGF production by peripheral blood mononuclear cells (PBMC) and T cells was studied using a new bioassay for BCGF activity. For this purpose, we established an Epstein-Barr (EB) virus-transformed B cell line KS-3.F10 that proliferates only in response to two B cell-specific BCGF, low-mol. wt BCGF (LMW-BCGF) and high-mol. wt BCGF (HMW-BCGF). PBMC from active SLE patients produced less BCGF when stimulated with phytohaemagglutinin (PHA) compared with controls. The decreased BCGF production by PHA-stimulated PBMC from active SLE reverted to control values when SLE became inactive. However, PHA-stimulated T cells from active SLE patients produced more BCGF compared with controls, whereas those from inactive SLE showed normal BCGF production. Spontaneous BCGF production by T cells was not observed in active SLE patients. These findings suggest that decreased BCGF production by SLE PBMC is due to excessive BCGF consumption by B cells in vitro and that SLE T cells produce large amounts of BCGF with appropriate immune stimuli in vivo to promote polyclonal B cell activation.     

Production of antineutrophil cytoplasm antibodies derived from circulating B cells in patients with systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. In certain systemic vasculitides, autoimmunity plays an important role with autoantibodies developing towards neutrophils, which are termed antineutrophil cytoplasm antibodies (ANCA). There is a growing body of evidence that T cells may contribute to the pathogenesis of ANCA-associated vasculitides. A system was set up to determine whether B cells require T cell help to produce antibodies in a peripheral blood lymphocyte (PBL) culture system enriched for B cells and dendritic cells (DC). As a control, tetanus toxoid (TT) antibody production was detected from individuals not recently immunized with tetanus vaccine when stimulated with TT antigen. Proteinase 3 (PR3) and myeloperoxidase (MPO) antibodies were produced from B cell and DC enriched cultures prior to the addition of antigen in some ANCA-positive patients with high ANCA titres, but not from patients with low ANCA titres or controls. PBMC from individuals recently immunized with tetanus vaccine were also maximally stimulated in that addition of antibody did not enhance antibody production. We conclude that this system supports a role for T cell help in the production of TT antibodies in individuals not immunized recently with tetanus vaccine. However, in patients with ANCA-associated vasculitis and controls recently immunized with tetanus vaccine, circulating B cells are apparently spontaneously producing autoantibody, possibly reflecting a system already maximally driven in vivo, and therefore masking underlying potential T cell-B cell collaboration. Such B cells may be less responsive to regulatory stimuli in vivo.     

In vitro induction of IgG anti-DNA antibody from high density B cells of systemic lupus erythematosus patients by an HLA DR-restricted T cell clone.

An HLA-DR restricted T cell clone (26G11) which recognized a lymphoid cell-derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti-DNA antibody production in DR-matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR-matched normal control subjects produced large amounts of IgM anti-DNA antibody, but did not produce IgG anti-DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR-matched patients with active SLE spontaneously produced a fairly large amount of IgG anti-DNA antibody, and the production was augmented by the T cell clone. Little IgG anti-DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD-B) and high density B (HD-B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti-DNA antibody was spontaneously produced by LD-B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti-DNA antibody was not spontaneously produced by the HD-B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti-DNA antibody-producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production.     

Stimulating and differentiation factors for human B lymphocytes in systemic lupus erythematosus.

T cells from 18 untreated SLE patients produced significantly more B cell growth factor (BCGF) than did those from normal subjects. Those from SLE patients with active disease produced significantly more than did those from patients with inactive disease. The response to BCGF of SAC-stimulated B lymphocytes from SLE patients was higher than that of B lymphocytes from normal individuals. Similarly preactivated B cells from five of seven SLE patients also proliferated upon the addition of interleukin 1 (IL-1) whereas those of normal subjects did not. Simultaneous addition of IL-1 and BCGF had a synergistic proliferative effect on B cells from two of seven SLE patients but not on any of the controls. Interleukin 2 (IL-2) had no proliferative effect in either SLE or normal B cells. Supernatant fractions from T cells of seven of 10 patients with active SLE and three of 10 with inactive SLE induced more IgG production by CESS cells than did those of normal subjects indicating a higher production of B cell differentiation factor by SLE T cells than by those of controls. Our findings may explain the reported preactivation and predifferentiation of peripheral blood B cells from SLE patients and give insight into the mechanisms leading to the production of autoantibodies in this disease.     

In vivo anti-nuclear antibodies in epithelial biopsies in SLE and other connective tissue diseases.

In vivo anti-nuclear antibody (ANA) was observed by direct immunofluorescence microscopy in epithelial cell nuclei in forty-four biopsies from thirty-three patients. The tissue containing the ANA was macroscopically normal in twenty-seven patients. The thirty-three patients with in vivo biopsy ANA included twenty-three with SLE, three with mixed connective tissue disease, two each with multi-system Sjögren''s syndrome, dermatomyositis, and progressive systemic sclerosis, and one with rheumatoid arthritis. Features of sicca syndrome were noted in seventeen patients. The patterns of the in vivo biopsy ANA in the thirty-three patients were speckled (21), homogeneous (6), nodular (2), and both speckled and homogeneous (4). Complement was not detected in the epithelial cell nuclei. Immunoglobulin(s) and/or complement were deposited along the dermoepidermal junction in thirty-two of the forty-four biopsies, and in dermal blood vessels in twenty-two biopsies. Each patient had serum ANA against rat liver substrate; twenty-seven had high titre ANA (1 in 1000 or greater). Elevated levels of DNA-binding were found in twenty patients (61%), but the level of DNA-binding did not correlate with the intensity of in vitro biopsy ANA staining. Serum antibody to ribonucleoprotein (RNP) was present in eight of the twenty-three patients tested (35%), all eight patients having clinical features of sicca syndrome. Hypocomplementaemia was found in thirteen patients (40%), all of whom had active SLE. In vivo biopsy ANA appears to be a real phenomenon of unknown aetiology, and not an artifact, which is found in some patients with active multisystem autoimmune disease, especially SLE.     

Localization of anti-nuclear antibodies on human metaphase chromosomes

A technique has been developed for the in vitro demonstration of anti-nuclear antibodies (ANA) in the sera of patients with systemic lupus erythematosus (SLE) utilizing human metaphase chromosomes in situ as substrate. The selective removal of histone, acidic protein, RNA and treatment with 2% formamide improves the binding of antibodies to DNA as demonstrated by fluorescein-conjugated and horseradish peroxidase-conjugated goat anti-human IgG. This technique is not only important because of the relative ease of demonstration of ANA but may be a valuable tool in the investigation of the pathogenesis of SLE.     

D-penicillamine: a modulator of anti-DNA antibodies production.

The effect of D-Penicillamine (DP) on the in vitro production of anti-DNA antibodies by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE) and from healthy individuals was studied. Anti-DNA antibodies were measured in culture supernatants using a sensitive microenzyme-linked immunoassay technique. The results of this investigation suggest that DP can act as an immunomodulator capable of potentiating or initiating anti-DNA antibodies synthesis as well as suppressing it. Although PBMC from both SLE patients and controls were responsive to this thiol compound, our results indicate that PBMC from patients with SLE were more susceptible to the enhancing effect of DP than did PBMC from controls. The cellular mechanism by which this drug can modulate anti-DNA antibodies production is discussed.     

Spontaneous production of antibodies to deoxyribonucleic acids in patients with systemic lupus erythematosus

In vitro spontaneous anti-DNA antibody production in patients with systemic lupus erythematosus (SLE) was examined. SLE lymphocytes produced IgG and IgM anti-DNA antibody from the third culture day, and reached a plateau on the seventh culture day. This anti-DNA antibody activity in 7-day culture supernatant was abolished by pretreatment of the lymphocytes with cycloheximide, suggesting de novo immunoglobulin synthesis was required for this spontaneous anti-DNA antibody production. Lymphocytes from patients with rheumatoid arthritis (RA) and other collagen diseases including progressive systemic sclerosis, polymyositis/dermatomyositis, and polyarteritis nodosa did not produce IgG and IgM anti-DNA antibody spontaneously, but SLE lymphocytes produced substantial amounts of IgG and IgM anti-DNA antibody spontaneously. Furthermore, active SLE produced a larger amount of IgG anti-DNA antibody than inactive SLE. We observed a significant negative correlation between the number of Ia+ T cells and IgG, but not IgM, anti-DNA antibody production. Furthermore, spontaneous IgG anti-DNA antibody production was elevated after pretreatment of SLE T cells with anti-Ia and complement, suggesting that Ia+ T cells in SLE bring about suppression of autologous B cells producing IgG anti-DNA antibody.     

B7/BB1 provides an important costimulatory signal for CD3-mediated T lymphocyte proliferation in patients with systemic lupus erythematosus (SLE).

Successful T cell activation via the T cell receptor (TCR)/CD3 complex requires at least one contact-dependent second signal delivered by costimulatory molecules, including the B7/BB1 molecule, that are present on antigen-presenting cells (APC). SLE is characterized by multiple complex lymphocyte abnormalities of undefined molecular origin. It is currently unclear whether an intrinsic defect of T cell or an underlying APC dysfunction is responsible for defective in vitro proliferation of T cells from patients with SLE. We planned the present experiments to ask whether the TCR/CD3-mediated and B7/BB1-costimulated T cell proliferation is normal in these patients. We used enriched T cell populations that were stimulated with an anti-CD3 MoAb in the presence of controlled quantities of functional B7/BB1 antigen. Freshly isolated T cells from 17 SLE patients (10 and seven patients with either active or inactive disease, respectively) and 11 normal individuals were cocultured with irradiated B7/BB1-transfected P815 cells or parental P815 cells in the presence of OKT3 MoAb at optimal and suboptimal concentrations for 2.5-7 days. Normal or SLE T cells responded similarly to stimulation via anti-CD3, in the absence of B7/BB1 antigen. A several-fold increase in T cell proliferation in the presence of B7/BB1 antigen was observed. Proliferation was inhibited in the presence of anti-B7/BB1 MoAb, but not with control MoAbs. Interestingly, dose-response curves and time kinetics of B7/BB1 costimulation were similar in T cells from patients with either active or inactive SLE at the time of study, and normal individuals. In addition, no differences in the IL-2 receptor release by T cells cultured under these conditions were observed between SLE patients and normal individuals. These results demonstrate that CD28 signalling is not intrinsically impaired in patients with SLE; further studies to investigate whether abnormal B7/BB1 expression is involved in the autoimmune process are needed.     

SLE病人外周血Bcl—2／JH基因重排检测的意义

应用聚合酶链反应（ＰＣＲ）方法检测３１例ＳＬＥ病人外周血单个核细胞（ＰＢＭＣ）Ｂｃｌ－２／ＪＨ基因重排现象和流式细胞仪间接双标记法分析其Ｔ（ＣＤ３）、Ｂ（ＣＤ１９）细胞Ｂｃｌ－２蛋白的表达。结果显示，ＳＬＥ病人Ｔ细胞Ｂｃｌ－２蛋白表达明显高于正常人（４２．９５％±２８．４７％对比９．９４％±４．９６％，Ｐ＝０．０００４），尤其以活动期ＳＬＥ病人为明显，而Ｂ细胞Ｂｃｌ－２蛋白表达与正常人之间并无统计学差异（７９．２１％±１０．６９％对比８１．９６％±６．９７％；Ｐ＝０．４６０２）。７例ＳＬＥ病人具有典型的Ｂｃｌ－２／ＪＨ基因重排（占２２．５８％），且均为ＳＬＥ活动期病人，其Ｔ细胞Ｂｃｌ－２蛋白表达明显高于无基因重排的ＳＬＥ病人，其Ｂ细胞Ｂｃｌ－２表达并无差异（Ｐ＞０．３９０５）。说明Ｂｃｌ－２／ＪＨ基因重排现象可见于ＳＬＥ，并与Ｔ细胞Ｂｃｌ－２蛋白高表达有关，表明细胞凋亡抑制基因Ｂｃｌ－２在ＳＬＥ发病机制中具有重要作用。     

Immunoregulation in glomerulonephritis, Henoch--Schonlein purpura and lupus nephritis.

Immunoregulation was examined in normal controls and in patients with immune complex glomerulonephritis and lupus nephritis (SLE) using OKT monoclonal anti-bodies against helper (OKT4) and suppressor (OKT8) T cell subsets. Functional studies assessed T cell control of in vitro immunoglobulin synthesis by cultured peripheral blood mononuclear cells (PBMC). IgG and IgA synthesis was measured in unstimulated, pokeweed mitogen (PWM) stimulated and PWM + concanavalin A (Con A) stimulated cultures. Patients with primary membranous nephropathy (MN) and mesangial IgA nephropathy (IgA GN) were found to have elevated T4/T8 ratios secondary to a deficiency of the T8+ subset. Patients with SLE had low T4/T8 ratios. B cell activation with high spontaneous immunoglobulin synthesis was present in cell cultures from patients with SLE, IgA GN and Henoch-Schonlein purpura (HSP). Defective Con A inducible suppression of in vitro immunoglobulin synthesis was found in SLE, HSP and to a lesser extent, primary MN. Functional Con A inducible suppressor defects correlated with elevated T4/T8 ratios only in patients with MN. All four disorders appear to share disturbances of cellular immune response with various degrees of defective immune suppression; however, it is not clear from these studies whether the defects are primary or secondary phenomena.     

Effects of immunoglobulin G from patients with systemic lupus erythematosus on human B cell function

We examined the effect of systemic lupus erythematous (SLE) sera and Ig fractions on IgG and IgM release by cultured normal peripheral blood mononuclear cells (PBMC) when these cells were preincubated with serum dilutions or Ig fractions. Increases in both IgM and IgG (P less than 0.001 and less than 0.01) in cultured cell supernatants were recorded when PBMC were preincubated with SLE serum dilutions. IgG but not IgM from SLE was found to stimulate PBMC to release IgG (P less than 0.01). Similar results were obtained when SLE IgG was preincubated with adherent cell depleted cells (ADC) or isolated normal B cell fractions. When normal PBMC were preincubated with SLE serum or IgG and subsequently stimulated with pokeweed mitogen (PWM), a relatively blunted IgG release was observed (P less than 0.05); however, IgM release was significantly increased (P less than 0.001). This effect was not observed when PBMC were preincubated with SLE IgM, normal serum dilutions, or normal Ig fractions. Relative blunting of PWM response after PBMC were preincubated with SLE IgG was not reversed in PBMC depleted of adherent cells, OKT8+, or OKT9+ cells. Depletion of PBMC of LeuM1 cells increased IgG release in response to PWM when cells had been preincubated with SLE IgG. SLE serum or Ig fractions did not induce B cell growth factor release by T cells. SLE IgG appeared to act directly on B cell enriched populations to release IgG; this was not associated with significant increase in thymidine uptake, or apparent lysis of cells.     

Inhibition of protein-kinase C in peripheral blood mononuclear cells of patients with systemic lupus erythematosus: effect on spontaneous immunoglobulin production.

Systemic Lupus Erythematosus (SLE) is a multisystem disease characterized by an increase in the spontaneous secretion of immunoglobulin (Ig) molecules, many of which are autoreactive. We have previously shown (Biochem & Biophys. Res. Comm. (1989) 161: 1319-1326) that normal human peripheral blood mononuclear cells (PBMCs) can be stimulated to secrete large quantities of Ig upon incubation with the protein kinase-C activator 1 oleoyl-2-acetyglycerol (OAG). Specific blockage of protein kinase C with the isoquinoline sulfonyl piperazine compound (H-7) inhibited the OAG-induced Ig production. In experiments reported here, PBMC of 5 patients with active SLE produced high levels of IgG spontaneously in culture. PBMC of 6 inactive SLE patients and 7 normal control subjects produced comparable low levels of IgG spontaneously. Pokeweed mitogen (PWM) stimulation of PBMC in inactive SLE and control groups, but not active SLE patients produced markedly enhanced IgG production. The lack of response to PWM stimulation in active SLE patients is likely due to inherent maximal stimulation of active SLE B-cells. In addition, we examined the ability of H-7 to inhibit both mitogen-stimulated (normal and inactive SLE) and spontaneous (active SLE) Ig production. In other experiments, we also examined the ability of the isoquinoline sulfonamide (HA-1004), a potent inhibitor of cAMP-dependent protein kinase to regulate mitogen stimulated and spontaneous Ig production in the patient groups indicated above. H-7 significantly inhibited PWM stimulated Ig production in normal (P less than 0.0001) and inactive SLE patients, (P less than 0.040) suppressing PWM stimulated levels to spontaneous levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

系统性红斑狼疮患者外周血单个核细胞中白细胞介素-10mRNA表达的研究

目的 探讨白细胞介素(IL-10)在系统性红斑狼疮(SLE)中的作用。方法 采用逆转录多聚酶链反应(RT-PCR)及酶联免疫吸附法(ELISA)测定40例SLE患者和20例正常对照组外周血单核细胞(PBMC)IL-10mRNA表达及IL-10自发分泌水平。结果 SLE患者PBMC自发分泌IL-10水平及其IL-10mRNA表达水平均显著高于正常对照组(P<0.01),其中SLE活动期明显高于非活动期(P<0.01),而非活动期又明显高于正常对照组(P<0.01)。结论 IL-10在SLE发病中起重要作用,PBMC分泌IL-10水平对SLE诊断和病情活动性监测有重要临床意义,拮抗SLE患者体内IL-10水平,将为SLE治疗开辟一条新途径。     

In vitro analysis of B lymphocyte function in uraemia.

We have investigated the immune responses in vitro of uraemic patients undergoing regular haemodialysis or continuous ambulatory peritoneal dialysis. Twenty-five healthy subjects were also studied as controls. In uraemic patients, the number of T and B lymphocytes were within the normal range, but proliferative responses to phytohaemagglutinin (PHA) were impaired. Spontaneous immunoglobulin plaque forming cell (PFC) responses by peripheral blood mononuclear cells (PBMC) from uraemic patients were significantly lower than those of healthy subjects. The PFC response of uraemic PBMC to the T cell independent polyclonal B cell activator (PBA) Epstein-Barr virus (EBV) was comparable to the response of the healthy subjects, indicating that uraemic B cells are still capable of synthesizing immunoglobulin. Pokeweed mitogen (PWM) induced PFC responses of uraemic PBMC were also normal, whereas the response to another T cell dependent B cell activator, Staphylococcus aureus Cowan I (SAC), was very low. Addition of indomethacin to PWM- and SAC-activated cultures of uraemic PBMC enhanced the PFC response to SAC, but had little effect on the PWM response. As full differentiation of B cells in response to SAC depends on helper T cells, we conclude that a defect in T lymphocyte function accounts for the reduced spontaneous and SAC induced production of immunoglobulin by uraemic PBMC. This defect may be mediated by an indomethacin-sensitive mechanism.     

In vitro IgM and IgM rheumatoid factor production in response to Staphylococcus aureus Cowan I: evidence for the role of Leu-2-positive T suppressor cells and radiosensitive T helper cells.

Staphylococcus aureus Cowan I (SAC) is a potent stimulus of B cell proliferation and differentiation, the latter being T cell-dependent. It has been suggested that immunoglobulin and IgM rheumatoid factor (RF) production in response to SAC involves radiosensitive T helper cells. We studied normal peripheral blood mononuclear cell (PBMC) cultures to assess the roles of radiosensitive T cells and Leu-2 positive suppressor cells in the cellular control of SAC-stimulated IgM and IgM RF responses. Depletion of Leu-2 positive T cells from reconstituted autologous PBMC cultures resulted in an increase in SAC-stimulated IgM production in the majority of individuals, implying the involvement of Leu-2 positive suppressor T cells in this system. Suppression by Leu-2 positive cells is less evident in the SAC-induced IgM RF response, suggesting qualitative differences between IgM and IgM RF SAC-stimulated responses in PBMC cultures from the same normal individuals. Irradiation (1000 rads) of the T cell-enriched subpopulation, either with or without Leu-2 positive cell depletion, resulted in statistically significant decreases in IgM and IgM RF production in response to SAC in reconstituted autologous cultures, providing further evidence of a Leu-2 negative radiosensitive sheep-cell rosetting cell active in in vitro SAC responses. In contrast, PWM-stimulated PBMC cultures showed almost exclusively increases in total IgM and IgM RF production when T cells were irradiated (1000 rads) before culture, consistent with the radiosensitive T suppressor cell involved in the in vitro immunoglobulin responses to PWM. The same five out of nine individuals produced IgM RF, under different culture conditions, in response to PWM and SAC, suggesting that the ability of an individual to produce IgM RF lies intrinsically within the B cell.