Molecular and functional analysis of dendritic cells in autoimmune disease

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by B cell hyperactivity and defective T cell functions including interleukin (IQ-2 production and proliferation. Any defect in T cell f unction, however, may be a consequence of an underlying functional aberration of the antigen presenting cell (APC) of which dendritic cells (DC), so far known, represent the most potent APC. In addition to the antigen specific signal, delivered by the T cell receptor (TCR)-CD3 complex, T cell activation -requires at least one costimulatory signal. Such a major costimulatory pathway involves the interaction between CD28 and B7 molecules. Little is currently known, however, about the role of costimulatory CD28-B7 interaction for the pathogenesis of SLE. Moreover, results regarding expression patterns of costimulatory molecules as well as functional capacities of APC in SLE patients are sometimes conflicting. This prompted us to set up this study in order to further define T cell:APC interactions in SLE patients. Using an in vitro model system will allow to perform detailed analyses on phenotypic characteristics of small proportions of peripheral blood DC as well as during a cytokine induced differentiation of DC from monocytes in SLE patients compared to healthy controls. Functional studies on the T cell stimulatory capacity of different DC populations performed in parallel will further allow to define molecular mechanism that enable DC to be crucially involved in the autoimmune response due to their specialized mechanism for antigen capture, uptake and processing. Such studies of phenotypic and functional DC characteristics therefore should enhance the knowledge about the pathogenic pathways involved not only in SLE but in autoimmune diseases in general and open new possibilities for prophylaxis and therapy.

Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease with a broad repertoire of clinical symptoms and serological alterations. The etiology of SLE still remains unclear. Beside genetic and infectious factors, environmental influences are considered to contribute to the pathogenesis of the disease. Numerous aberrations of the immune system, especially of T lymphocyte function have been described in patients with SLE (1, 2). For optimal proliferation T lymphocytes require two signals (3). The first signal is delivered by binding of the antigen (Ag) to the T cell receptor (TCR). Therefore the Ag has to be processed by an Ag presenting cell (APC) and presented in conjunction with major histocompatibility complex (MHC) class II molecules on the surface of APC. Among different APC populations, dendritic cells (DC) have been shown to be very potent in the activation of naive T cells (4, 5). During the recent years several studies showed that patients with SLE exhibit altered APC function (6, 7, 8). But results are contradictory. We therefore wanted to compare the phenotype and function of DC derived from patients with SLE with cells from healthy controls. Material and Methods Lymphocytes and monocytes were isolated from the peripheral blood by density centrifugation (over Ficoll) and cell sorting by magnetic beads conjugated antibodies (MACS). Afterwards cells were characterized by immunofluorescence and flow cytometry (FACS). Furthermore accessory cell function was tested in vitro using autologous and allogenic mixed lymphocyte reactions. These stimulations were performed in different cell ratios (T cells:DC 2:1, 6:1, 18:1, 54:1). Autologous T cell stimulation was performed with tetanus toxoid and KLH. To generate DC, freshly isolated monocytes were cultured for 8 days in the presence of cytokines (GM-CSF and IL-4). These "immature" DC were analyzed and used to stimulate T cells as described above. Subsequently "immature" DC were stimulated by LPS for another 2 days. The "mature" DC derived from these cultures again were tested and analyzed in similar experiments. We acquired results for monocytes and DC from healthy controls and SLE patietns for days 0, 8 and 10. Results Restimulation assays with DC derived from patients with SLE revealed a decreased costimulatory capacity compared with cells from healthy subjects. The differences were more evident at T cell/DC ratios between 6:1 to 18:1. This has been observed for both, immature (after 8 days of culture) and mature (after 10 days of culture) DC. Moreover, the surface expression of accessory molecules (CD 80, CD83, CD54, and HLA-DR) was significantly decreased on DC cultured for 10 days. No differences were observed for CD86 and CD40 between patients and healthy controls. Furthermore in vitro differentiation from monocytes to DC seems to be disturbed in patients with SLE compared to healthy controls. After 8 and 10 days of cuture, percentages of DC expressing CD1a were significantly lower in the SLE group. Discussion As shown recently (9), our results hint at a defect of APC function in autoimune diseases, especially in SLE. The phenotypical differences of DC derived from SLE patient and healthy individuals shown here are not able to fully explain the functional alterations of these cells. Perhaps other factors, i.e. altered cytokine production, may contribute to these phenomenons. Further investigations have to be done to answer these questions.