The soluble CD40 ligand sCD154 in systemic lupus erythematosus

Abstract

We found that the plasma of patients with active systemic lupus erythematosus (SLE) could induce a human B-cell line (Ramos) to express high levels of immune accessory molecules that are commonly found on blood B cells of patients with active SLE. The ability of SLE plasma to induce such phenotypic changes could be abrogated by neutralizing antibodies specific for the CD40 ligand (CD154) but not by antibodies to TNF-alpha. Immunoprecipitation studies with anti-CD154 identified a 20-kDa protein in the plasma of SLE patients with active disease, but not in plasma of normal donors, indicating that such plasma contained soluble CD154 (sCD154). Using a quantitative ELISA method, we found that the plasma of patients with active disease had levels of sCD154 that were significantly higher than those found in plasma of normal donors. Levels of CD154 transcripts in SLE blood lymphocytes correlated with the relative concentrations of sCD154 found in SLE plasma. Furthermore, plasma levels of sCD154 correlated with the titers of anti-double-stranded DNA autoantibody and with clinical disease activity. These studies indicate that sCD154 of patients with SLE may act as a functional ligand for CD40 that is associated with SLE disease activity.

Figures

Figure 1

Changes in surface antigen phenotype of Ramos B cells after culture in human plasma depleted of fibrinogen. Ramos B cells were cultured in media supplemented with 50% human plasma either without (a and b) or with (c and d) a neutralizing mAb specific for CD154. The cells were examined for expression of CD54 (a and c) or CD95 (b and d) using flow cytometry. The histograms depict the relative cell number and the logarithmic fluorescence intensity. Open histograms represent staining of Ramos cells with an isotype control mAb. Hatched and filled histograms depict the staining of Ramos cells with antigen-specific mAb after culture in plasma from a control donor (NOR-plasma) or patient with SLE (SLE-plasma), respectively.

Figure 2

Immunoblot analyses of sCD154 isolated from culture supernates of human CD154–transfected HeLa cells (a and b) or human plasma samples (c). Cell-free supernatants (SUP), or plasma from a control donor (H1) or each of 2 SLE patients (S1 and S2), were absorbed onto anti-CD154 mAb–coupled Sepharose beads. The affinity-bound material was eluted, and then subjected to SDS-PAGE and immunoblot analysis with anti-CD154 antisera specific for the extracellular (a and c) or cytoplasmic (b) domain of CD154, respectively. The left lanes in a and b (labeled MW) are proteins of known molecular size that serve as standards. To the left of each panel are lines indicating the migration distance of each of these proteins, along with its molecular size. The arrows at the right of each panel indicate the expected migration distance of sCD154.

Figure 3

ELISA to detect sCD154 concentrations in human plasma. (a) A standard curve for serial dilutions of a recombinant sCD154 placed into separate wells of a microtiter plate precoated with anti-CD154 mAb (TRAP1). Bound recombinant sCD154 was detected using a non–cross-reacting, biotinylated anti-CD154 mAb (M90). The mean absorbance of triplicate wells is indicated on the ordinate, and the logarithmic concentration (picograms per milliliter) of the recombinant sCD154 is provided on the abscissa. (b) Indicated are the measured concentrations of sCD154 in plasma isolated at various times after phlebotomy from whole blood kept at room temperature (filled circles) or at 4°C (open circles). The calculated concentration of sCD154 is listed on the ordinate (picograms per milliliter), and the time after phlebotomy prior to plasma separation is listed in hours on the abscissa. Each point represents the mean value of triplicate wells, and the error bars indicate the SE. (c) The concentrations of sCD154 (picograms per milliliter) that were detected in the plasma of each of 21 control donors (open circles) or 26 patients with SLE (filled circles) are indicated. Each point represents the concentration found in a single donor. The bar indicates the mean concentration for the healthy and SLE groups. The numbers indicate the mean concentration for each group ± SD about the mean. A dotted line represents the mean + 3 SD of sCD154 in control donors. The P value is calculated for the difference between the means of the 2 groups (P < 0.0001).

Figure 4

Comparison of the numbers of CD154 transcripts per cell with the measured sCD154 plasma concentration or the intensity of CD154 surface membrane expression on blood mononuclear cells of patients with SLE. The numbers of CD154 transcripts per freshly isolated mononuclear cells of 10 patients with SLE are indicated on the abscissa. (a) Each dot represents the mean plasma concentration of sCD154 detected for each patient, as indicated on the ordinate (picogram per milliliter). These points are plotted against the numbers of CD154 transcripts per cell detected in the mononuclear cells isolated from the whole-blood sample from which each plasma sample was derived. The line depicts the best-fit correlation between the levels of CD154 transcripts per cell and the plasma concentration of sCD154 (Pearson r = 0.779, P = 0.01). (b) Each dot represents the intensity of CD154 membrane expression on the blood mononuclear cells of each patient, indicated on the ordinate as the MFIR of cells stained with anti-CD154 mAb and a control mAb of the same isotype. The line depicts the best-fit correlation between the levels of CD154 transcripts per cell and the intensity of CD154 surface membrane expression (Spearman r = 0.194, P = 0.584).

Figure 5

Relationship between sCD154 plasma concentration and SLE disease activity. (a) Described are the measured sCD154 plasma concentrations of control donors (open circles in column 0), SLE patients with low disease activity (SLEDAI between 2 and 9; shaded circles in column labeled 2–9), and patients with active SLE (SLEDAI ≥10; filled circles in the right column labeled ≥10), as indicated on the abscissa (nanogram per milliliter). Each dot represents the measured plasma concentration for an individual subject. The P values, indicating the differences between the mean values for each group, are indicated at the top of the figure. The difference between the inactive and active SLE groups is statistically significant (P < 0.0001). (b) Described are the relationships between measured plasma concentrations of sCD154 (as indicated on the ordinate in nanogram per milliliter) and the anti-dsDNA Ab levels in each of 23 SLE patient samples, as indicated on the abscissa (IU/mL). The line represents the best-fit correlation between the plasma concentrations of sCD154 and the anti-dsDNA Ab levels (Spearman r = 0.758, P < 0.001). Four of the values represent measurements made of serial samples obtained from the same patient (filled squares labeled 1, 2, 3, and 4). Square 1 is the initial sample taken before immune suppressive therapy. Samples for squares 2, 3, and 4 were obtained from the same patient 4, 8, and 17 weeks later, respectively.