Keratinocytes from patients with lupus erythematosus show enhanced cytotoxicity to ultraviolet radiation and to antibody-mediated cytotoxicity

Abstract

Keratinocyte cytotoxicity is an important component of the immunopathology of photosensitive lupus erythematosus, and antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be an important mechanism by which autoantibodies, especially those specific for SS-A/Ro, can induce keratinocyte damage in models of photosensitive lupus. We provide further evidence that keratinocytes from patients with photosensitive lupus show significantly greater ultraviolet radiation (UVR)-induced cytotoxicity, and that ADCC of these targets is especially enhanced by autologous patient's serum or by anti-SS-A/Ro+ sera. Keratinocytes from normal uninvolved skin of 29 patients with cutaneous lupus erythematosus (LE) were grown in cell culture and tested as targets in cytotoxicity experiments in vitro. Cultured keratinocytes from patients with systemic lupus erythematosus (SLE) and subacute cutaneous lupus erythematosus (SCLE) showed significantly greater cytotoxicity following UVR treatment than did keratinocytes from normal adult controls or from neonatal foreskins (P < 0.01). The same cultures also showed greater UVR-induced binding of IgG from fractionated anti-SS-A/Ro+ preparations. ADCC experiments were also performed using keratinocytes cultured from patients with SLE, SCLE, discoid lupus erythematosus (DLE), and normal controls. When keratinocytes were incubated in autologous serum plus a standard mononuclear cell effector population, the percentage of ADCC observed was significantly greater in cultures containing keratinocytes and sera from the SLE and SCLE patients (P < 0.001). When cultured keratinocytes were added to different IgG antibody probes, plus standard mononuclear effector populations, greater ADCC was seen using the anti-SS-A/Ro probe and keratinocytes from patients with SLE or SCLE. With normal human neonatal keratinocyte targets, the anti-SS-A/Ro probe induced greater ADCC than that seen with anti-ssDNA or normal human serum. We have shown that keratinocytes from patients with some forms of lupus erythematosus (SLE and SCLE) show greater cytotoxicity in vitro when irradiated with UVR, and greater susceptibility to ADCC whether the antibody source is their own serum or an anti-SS-A/Ro probe.

Fig. 1

Autologous combinations of targets and serum from lupus patients but not normal controls enhance antibody-dependent cell-mediated cytotoxicity (ADCC) of irradiated keratinocytes. In this assay ADCC (cytotoxicity percentage) was measured using an acridine orange/ethidium bromide assay in 24 h cytotoxicity experiments. Each circle represents an individual data point, and the lines and brackets indicate the mean ± s.d. Each data point is an ADCC experiment containing autologous cultured keratinocytes (T) and sera (A) from one subject plus a standard homologous mononuclear effector (E). The keratinocytes and sera were from patients with systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus (SCLE), or discoid lupus erythematosus (DLE) or from normal controls. The keratinocytes were irradiated with 100 mJ/cm² of UVB light 24 h before addition of sera and effectors. The percentage cytotoxicity was determined by the following formula comparing different combinations of T, A and E. ADCC of irradiated targets = (T_UVRAE − T_UVRE) − (TAE − TE), where T_UVR are targets irradiated with UVB 24 h before the experiment was performed, and T are keratinocytes which were sham-irradiated. Irradiated targets incubated with anti-SS-A/Ro antibody did show some cytotoxicity without added mononuclear effectors, but it represented only a fraction of the cytotoxicity observed with antibody plus effectors (data not shown). Statistical significance of different comparisons is indicated by P values.

Fig. 2

Purified IgG from anti-SS-A/Ro pooled sera significantly increase the level of antibody-dependent cell-mediated cytotoxicity (ADCC) against normal neonatal keratinocyte targets. In this assay, ADCC of normal neonatal foreskin keratinocytes was measured by ethidium bromide/acridine orange assay. Each circle represents one experimental point, each line and bracket shows the mean ± 1 s.d. of the points derived from three experiments. Twenty-four-hour ADCC experiments were performed using combinations of target (T), antibody (A) and effectors (E). The ADCC experiments were performed using normal neonatal keratinocyte cultures (T) and normal control mononuclear effectors (E). The antibodies used to induce ADCC were present in pooled sera from autoimmune patients specific for anti-SS-A/Ro or anti-ssDNA, or from normal human sera. The IgG fractions from the sera were obtained by fractionation with protein-A Sepharose. The specificity of the anti-SS-A/Ro fraction was verified by immunoblot and by blocking with purified SS-A/Ro. The targets were either irradiated keratinocytes (T_UVR) exposed to 100 mJ/cm² of UVB light 24 h prior to combination with antibody plus effectors, or non-irradiated keratinocytes (T). ○, Percentage cytotoxicity of TAE − TE; •, percentage cytotoxicity of T_UVRAE − T_UVRE. Irradiated targets incubated with anti-SS-A/Ro antibody did show some cytotoxicity without added mononuclear effectors, but it represented only a fraction of the cytotoxicity observed with antibody plus effectors (data not shown).