The immunological personality of close relatives of SLE patients.

Immunological abnormalities seen in relatives of patients with autoimmune disorders can be useful in understanding the pathogenesis of the disease since, unlike in patients, they cannot result from the disease process or drug treatment. In this article we present a brief overview of our studies of the basic immunological status of close relatives of SLE patients. We looked at blood levels of IgG, IgM and antibodies to double-stranded DNA, as well as at NK cell numbers and cytotoxic activity and the levels of NKT, B and T cells. As many as 60% of relatives showed one or more abnormalities in these assays. Most notably there were increased levels of IgG in male and female relatives and a reduction of IgM in females. IgG correlated inversely with NKT cell numbers adding strength to the concept that the presence of IgG autoantibodies in patients is due to impaired regulation by NKT cells. IgM, on the other hand, correlated inversely with NK cells which may thus have a role in bringing about the reduced IgM seen in some patients. Immunological abnormalities were found to be more often associated with parents and offspring of patients than with their siblings, pointing to the involvement of environmental or epigenetic influences in lupus pathogenesis.

Serendipitous evidence of T lymphocyte activation in close female relatives of patients with systemic lupus erythematosus.

A well-recognized characteristic of the autoimmune disease, systemic lupus erythematosus (SLE), is the high level of activated T cells present in the blood. Because of the increased size and granularity of activated T cells, in flow cytometry one might expect to find increased numbers of cells falling outside a standard light-scatter lymphocyte gate, and indeed we now report that the percentage of T lymphocytes in the gate (% TiG) was below the normal range in 23 of 58 (40%) female patients because of increased scatter values. However, the surprising additional observation was made that 18 of 30 (60%) female first-degree relatives of the patients also fell below the normal % TiG range, suggesting the presence of T cell activation in these relatives. This view is strengthened by the strong inverse correlation between plasma total immunoglobulin G(IgG), which was raised in some relatives, and % TiG, as T cell activation is a requirement for IgG production. Conversely, there was no correlation with IgM, which has no comparable link with T cell activation. While a definitive interpretation must await the demonstration of activation antigen expression in relatives, these findings suggest the existence of a T cell activation trait, not harmful in itself, which, however, contributes to the development of disease in patients with SLE.

Natural killer cell activity in families of patients with systemic lupus erythematosus: demonstration of a killing defect in patients.

Natural killer (NK) cell cytotoxic activity and cell frequency, expressed as a percentage of total lymphocytes, have been determined in peripheral blood mononuclear cells from first-degree relatives of patients with systemic lupus erythematosus (SLE), the patients themselves, a group of rheumatoid arthritis (RA) patients and controls. Low levels of killing activity relative to controls were found in some members of all groups with the extent of depression falling into two ranges. Moderate reductions were seen in female (3/31, 10%) and male (4/14, 29%) relatives of SLE patients, female (12/60, 20%) and male (3/4, 75%) SLE patients and female RA patients (6/17, 35%). A more profound depression of killing activity was confined to other female SLE patients (15/60, 25%). There were strong correlations in all groups between killing activity and percentage of NK cells, but analysis of the ratio of these parameters and studies with purified preparations of NK cells suggest that the reduced activity in SLE frequently involves a defect in the killing capacity of the individual cells in addition to the reduced levels of NK cells. Azathioprine (AZA), which was used in treatment of 12 SLE patients, was invariably associated with low values of killing activity. It appears to substantially reduce the percentage of NK and B cells in an action unconnected with the NK cell abnormalities associated with SLE. The finding of low killing activity in relatives and a correlation between their activity and that of their patients support the view that NK cell deficiency is a genetic determinant of SLE. NK cells in SLE may produce insufficient levels of cytokines required for the regulation of IgG production.

Counter-proliferative effects of nucleosomal antigens in cultures from lupus patients.

Blood mononuclear cells from 20 lupus patients were cultured in the presence of nucleosomal antigens to determine whether they induce lymphocyte proliferation. The predominant effect seen, however, was one of inhibition of the background proliferation. Such inhibition was rare with cells from female or male controls. Nucleohistone (NH), crude histone and enriched preparations of histones H2A/H4, H2B and H3 showed this effect in approximately one-third of patients, but H1 and single-stranded (ss) DNA had no such activity. Double-stranded (ds) DNA may show this inhibitory action, but further tests are required. ssDNA was the only antigen that showed evidence (two patients) of disease-related stimulation of proliferation. Histones and NH induced proliferation in many subjects but the strongest responders were controls. Patients responded poorly to tuberculin PPD but gave an exceptionally strong proliferative response to pokeweed mitogen. It is suggested that the inhibition of background proliferation in patients is a consequence of the interaction of nucleosomal antigens with sensitised T cells. If T cell sensitisation to histones is an important factor in the development of lupus, the disease may be preventable in those at risk by inducing tolerance to the appropriate peptides.

Spontaneous immunoglobulin-producing capacity of cultures from lupus patients and normal donors following depletion of cells expressing CD19 or CD38.

Cells spontaneously secreting IgG or IgM (ISC) are present at a high level in the blood of patients with systemic lupus erythematosus (SLE). By use of magnetic-bead techniques, mononuclear cells from such patients and healthy donors were fractionated according to expression of CD19 or CD38 and the cell fractions were then cultured in the absence of added mitogen/antigen for 5/6 days. Supernatant IgG and IgM were determined and, in addition, in the CD38 experiments ISC were enumerated both before and after culture. Much of the immunoglobulin-producing capacity of unfractionated cells (UFC) from both donor groups was recovered in the CD19- fraction, and no immunoglobulin was produced by CD19+ cells suggesting, unexpectedly, that ISC were not expressing CD19. By contrast, CD38 fractionation resulted in nearly all ISC passing to the CD38+ fraction which produced levels of immunoglobulin approaching 50% that of UFC. On culture of CD38- cells there was a build up in the number of IgG and IgM ISC, this being particularly striking in the controls with numbers well in excess of those in UFC. Not all these new ISC became CD38+, but the maturation process was more efficient in the SLE patients. The possibility is discussed that the spontaneous response in the CD38- populations is due to removal of CD38+ natural killer (NK) cells. Removal of ISC that are present preculture is a helpful initial step in studying ISC generation in the disease.

B lymphocyte hyperactivity in families of patients with systemic lupus erythematosus.

Blood cells spontaneously secreting IgG and IgM and the level of plasma immunoglobulins and antibodies to dsDNA, ssDNA and influenza virus haemagglutinin have been determined in families of patients with SLE, in 'normal' families and groups of 'normal' individuals. IgM values were consistently higher in females than in males. About one in three of healthy blood relatives gave values in excess of the sex-matched control range in one or more of these test, particularly notable being raised values of IgG anti-dsDNA and total IgG shown by female relatives. High-scoring relatives were more likely to be offspring or parents than siblings of patients, suggesting, together with evidence from a spouse group, the involvement of environmental as well as genetic factors in these families. Correlation analysis between the various assays in the different groups showed a clear distinction between the female control group, where there were no associations, and the female relative group where there were strong associations, including a significant correlation between IgM and IgG antibodies to dsDNA. The male groups produced a more variable picture but the patients gave a remarkably consistent pattern of moderate positive associations. Pokeweed mitogen induced a higher level of IgG production in blood cells of relatives than in controls. These findings are suggestive of a breakdown in relatives of normal antibody regulation. Investigation of immunological abnormalities in family members provides a powerful tool for the analysis of a complex disease.

Inhibition of mouse spleen cell activity by organotin compounds: effect of attachment of a maltose residue to the organotin group.

Studies are reported on the inhibition of DNA synthesis and the lowering of cell viability caused by bis(tributyltin) oxide in mouse spleen cells cultured in the presence and absence of the B-lymphocyte mitogen, bacterial lipopolysaccharide. When a maltose residue is introduced into the organotin compound these toxic effects are increased. It is suggested that the maltose residue facilitates entry of the organotin compound into the cells.

Does mitogen-induced antibody production by normal blood cells mimic spontaneous production in lupus?

Blood cells from patients with systemic lupus erythematosus (SLE) showed a raised level of spontaneous IgG production that included antibodies to DNA and to common environmental antigens (influenza virus haemagglutinin, adenovirus hexon and mannan from Candida albicans). In contrast, no IgG antibody was produced against an antigen not normally encountered in the UK (egg antigen from Schistosoma mansoni) or a self-antigen not generally associated with SLE (thyroglobulin). IgM production was raised to a lesser extent and only antibodies to DNA were detected. When normal cells were stimulated with pokeweed mitogen or S. aureus organisms, the specificity pattern of IgG production was similar to that described above for SLE with the major exception of the absence of IgG anti-DNA. IgM antibodies to DNA and all the other antigens were detected, but the specificity of the IgM ELISA assays for the protein antigens needs further clarification. The activity of IgM anti-DNA relative to total IgM was far greater in the SLE system. These results provide further evidence that a response to self-antigen is required for production of pathogenic IgG autoantibodies in SLE.

Spontaneous antibody-secreting cells against DNA and common environmental antigens in systemic lupus erythematosus.

Cells spontaneously secreting IgG or IgM antibodies to DNA or to common environmental antigens--influenza virus haemagglutinin, adenovirus hexon and mannan from Candida albicans--have been enumerated by ELISA spot in blood from patients with systemic lupus erythematosus (SLE) and normal donors. Mean values were raised for all antigens in the disease, with those for DNA being no greater than for the other antigens. In normal donors, levels of IgM-secreting cells were similar for DNA and the environmental antigens whereas virtually no IgG anti-DNA secreting cells were found. When results were expressed relative to total numbers of IgG or IgM-secreting cells, the differences between the groups disappeared or were greatly reduced in all systems except IgG anti-DNA. These findings are consistent with a requirement for both polyclonal activation and a self-antigen response in the production of IgG autoantibodies in SLE.

Tuberculin sensitivity and an interaction of leucocyte and plasma factors involving fibrinogen.

Lysates of blood leucocytes from strong tuberculin-positive reactors contained smaller particles of insoluble protein-rich material than did similar preparations from negative donors. The phenomenon was dependent on the presence of small quantities of plasma in the original leucocyte suspensions: when this was removed or replaced by serum both groups of donors produced particles of the smaller size. When the experiments were repeated in the presence of 125I-fibrinogen, larger particle size was found to be associated with an increased proportion of radioactivity becoming firmly bound to the insoluble material. These findings are likely to reflect differences in the levels of procoagulant, proteolytic enzymes or associated activities in the cells or plasma of the two groups.

B lymphocyte activation in systemic lupus erythematosus: spontaneous production of IgG antibodies to DNA and environmental antigens in cultures of blood mononuclear cells.

IgG antibodies to DNA, influenza virus haemagglutinin (HA), adenovirus hexon (HX) and mannan from Candida albicans (MN) have been determined in supernatants from 2-day unstimulated cultures of peripheral blood mononuclear cells from SLE patients and controls. Mean values were much higher in the SLE group, with from 20% (MN) to 85% (DNA) of patients giving values above the normal range. Although a significant correlation was observed between anti-DNA and anti-HA production, anti-HX and anti-MN showed no such correlations. The specificity of the ELISA assays was demonstrated by inhibition tests. It is concluded that a selective form of polyclonal activation in SLE results in the production of antibodies to foreign as well as to self antigens.

Leucocyte migration inhibition in diphtheria toxoid hypersensitivity.

A donor who was highly reactive to diphtheria toxoid (DT) in delayed hypersensitivity and in lymphocyte transformation showed scant evidence of antigen-induced inhibition in the direct leucocyte migration agarose test. Other donors, weak or negative to DT in skin test and transformation, did show evidence of inhibition. Although the migration test is useful in assessing cellular reactivity to tubercular antigen, these results question its suitability for DT.

Evaluation of an ELISA system for determination of class-specific antibodies to native and denatured DNA in man.

Enzyme-linked immunosorbent assays (ELISA) have been set up for determination of plasma IgG and IgM antibodies to native (n) and denatured (d) DNA. Normal male and female donors generally gave low values in the assays for IgG; IgM control values were higher, particularly in females. Mean values for patients with systemic lupus erythematosus (SLE) were greatly raised in all four categories of assay in relation to the control (female) group. Levels of IgG anti-nDNA in SLE correlated well with a standard diagnostic test (Farr), and this ELISA assay was more successful than Farr in discriminating between patients and normal females. No such correlation with Farr was found for IgM anti-nDNA. Correlations were found in SLE between levels of antibodies to nDNA and dDNA. Inhibition tests--including those with a plasmid DNA preparation containing no single-stranded regions--showed that most of the IgG antibodies determined in the 'native' assay were able to bind to nDNA and dDNA with comparable avidity, whereas most of those reacting in the 'denatured' assay could only bind dDNA. The former antibodies were probably directed against shared determinants on the deoxyribose-phosphate backbone and the latter against base-dependent structures not exposed in nDNA. Inhibition results for IgM assays were similar, though the predominance of antibodies specific for dDNA appeared less marked. ELISA assays could well prove more useful than established methods in diagnosis and monitoring of SLE and other diseases.

The effect of hydrocortisone on PWM and LPS-induced immunoglobulin production in blood leucocytes from patients with systemic lupus erythematosus.

The effect of hydrocortisone (HC) has been investigated on PWM and LPS-induced immunoglobulin (Ig) production by blood mononuclear leucocytes (MNL) from normal donors and patients with systemic lupus erythematosus (SLE). In the absence of HC, PWM stimulated Ig production in normal but not in SLE cells. In the presence of HC, the normal PWM response was greatly enhanced and a strong response was now seen in SLE. Spontaneous Ig production, which was higher for SLE-MNL, was not greatly affected by HC. A response to LPS was observed in both normal and SLE-MNL in the absence of HC and HC was now inhibitory, confirming that the mode of action of the 2 mitogens is different. It seems likely that HC acts by suppressing cells which influence mitogen responses, and the possibility that the principal target, at least in SLE-MNL, is the monocyte is discussed. The stimulatory effect of HC could be useful in the study of antigen-induced Ig production in SLE.

Dual action of leucocyte dialysates and of thymosin on the recovery of sheep-cell-rosetting capacity in trypsinized human lymphocytes.

Trypsinization of human blood lymphocytes abolishes their capacity to form rosettes with sheep erythrocytes (E-rosettes) and this is regained in part on incubation of the cells at 37 degrees C for 3 hr. The recovery of rosetting capacity was found to be accelerated in the presence of dialysable extracts of human leucocytes (DLE) or bovine thymosin fraction 5 (THFV). For both DLE and THFV two types of effect were demonstrated. At lower concentrations the stimulation of recovery was dependent on the presence of the agent during incubation and it presumably comes about through an effect on the metabolic process required for regeneration of the E-receptors. At higher concentrations another mechanism is apparent since the agents were now effective when added after incubation. This last phenomenon is wholly dependent on prior incubation of the trypsinized lymphocytes in medium alone and it probably involves attachment of components of DLE and THFV to incompletely recovered cells, thereby providing a more favourable charge environment for E-rosette formation. A similar process of adhesion-promotion may be occurring in certain in-vitro tests with THFV which are carried out on lymphocytes from immunodeficient patients. On the other hand, it is the other mechanism, that of metabolic action, which is likely to be the predominant consideration in relation to treatment of such patients with DLE or THFV.

Effects of human transfer factor on the migration of guinea-pig macrophages: is there an antigen-specific activity?

Dialysable transfer factor (TF) prepared from the buffy-coat cells of tuberculin-positive human donors exerted antigen (PPD)-dependent inhibition of migration of guinea-pig peritoneal exudate cells (PEC) provided that migration of the cells was not strongly affected by PPD alone. TF from tuberculin-negative donors did not do this. The effect could be better demonstrated with tuberculin-sensitive than with normal PEC. Differences in the actions of 'tuberculin-positive' and 'negative' TF may also be seen in the absence of antigen. In a similar but more restricted series of experiments with diphtheria toxoid (DT) as antigen, DT-dependent inhibition was observed only with 'DT-positive' TF. The findings concerning antigen-dependent inhibition in both the tuberculin and toxoid systems are compatible with the concept of an antigen-specific TF, but it is argued that they should not be taken as strong evidence of such specificity. In the tuberculin system the results suggest an alternative explanation, namely that 'tuberculin-positive' TF contains a higher level of a non-specific activity. Whether specific or not, the antigen-dependent activity probably involves a stimulatory action on antigen-induced lymphocyte activation leading to enhanced production of macrophages migration inhibition factor, and it could be related to the 'transfer' phenomenon in vivo.

An investigation into the antigen-specificity of transfer factor in its stimulatory action on lymphocyte transformation.

Dialysable transfer factor (TF) was prepared from the buffy-coat cells of donors with known cell-mediated reactivity to tuberculin (PPD), streptococcal protein (SKSD) and diphtheria toxoid (DT). The effect of such preparations on the transformation by these antigens of lymphocytes from tuberculin-negative donors was investigated. Transformation was determined as incorporation of tritiated thymidine. The concentrations of SKSD and DT were adjusted for different lymphocyte donors so as to give, in the absence of TF, a low index of transformation (less than 10-fold) comparable to that obtained with PPD. TF from tuberculin-positive donors stimulated antigen-induced transformation by on average approximately 2-fold whereas TF from tuberculin-negative donors generally had little effect. This was so not for PPD as antigen but also for SKSD and DT, and sensitivity of TF donor to SKSD of DT was not a determining factor. TF also frequently increased background transformation in the absence of antigen. Although a small effect, this ability tended to reflect the activity of TF in the presence of antigen. It is concluded that neither the whole nor any significant part of this enhancement of transformation can be ascribed to an antigen-specific factor. Tuberculin-positive donors apparently yield a higher level of non-specific factor and possible reasons for this are discussed. The factor active in transformation may be responsbile for the TF phenomenon in vivo.