Patients with active SLE often have an ongoing production of IFN-alpha. We therefore searched for an endogenous IFN-alpha-inducing factor (IIF) in SLE patients and found that their sera frequently induced production of IFN-alpha in cultures of peripheral blood mononuclear cells (PBMC) from healthy blood donors, especially when the PBMC were costimulated with the cytokines IFN-alpha2b and granulocyte-macrophage colony-stimulating factor (GM-CSF). The phenotype of the IFN-alpha-producing cells (IPC) as determined by flow cytometry corresponded to that of the natural IPC, resembling immature dendritic cells. The IIF activity in SLE sera was sometimes as high as that of a virus and was present especially in patients with active disease and with measurable IFN-alpha levels in serum. The IIF had an apparent molecular weight of 300-1000 kD and appeared to consist of both immunoglobulin and DNA, possibly being immune complexes. This endogenous IFN-alpha inducer may be of pathogenic significance, since a reported occasional adverse effect of IFN-alpha therapy in patients with non-autoimmune disorders is development of anti-dsDNA antibodies and SLE.
Systemic lupus erythematosus (SLE) patients have increased levels of interferon-alfa (IFN-alpha) in the circulation but a reduced number of functionally intact natural IFN-alpha producing cells (IPC) in peripheral blood. In search for tissue localisation of activated IPC, we investigated skin biopsies from SLE patients for the occurrence of such cells. Eleven SLE patients with inflammatory skin lesions and six healthy controls were biopsied. An immunohistochemical technique (IH) and in situ hybridisation (ISH) were used to detect intracellular IFN-alpha protein and IFN-alpha mRNA, respectively. In all 11 biopsies from SLE lesions, a high number of IPC were detected by IH. In the nonlesional SLE biopsies we could also demonstrate IPC in 10/11 patients. In 6/11 SLE patients, IFN-alpha mRNA containing cells could be detected in the specimens. A low number of IPC were detected in 1/6 healthy controls by IH, but no ISH positive cells were seen. Our results demonstrate that SLE patients have active IPC in both dermal lesions and in noninflammatory skin. A recruitment of IPC from blood to peripheral tissues may explain the low number of circulating natural IPC in SLE patients. Because the type I IFN system is involved in the SLE disease process, these results are of interest for the understanding of the pathogenesis in SLE.
The relationship between the so-called natural interferon-alpha (IFN-alpha) producing cell (IPC), stimulated to produce IFN-alpha by herpes simplex virus type 1 (HSV), and of dendritic cells (DC) in peripheral blood leucocytes was investigated. The simultaneous expression of cell surface antigens and intracellular IFN-alpha in the HSV-stimulated IPC (HSV-IPC) was examined by flow cytometry (FCM). The HSV-IPC were infrequent, < 0.3% of the mononuclear leucocytes, and with homogeneous light scatter characteristics. The HSV-IPC were confirmed to lack leucocyte lineage specific markers, and to express CD4, CD36 and HLA-DR. Furthermore, they expressed high levels of CD44, CD45RA and CD45RB, and lower levels of CD40, CD45R0, CD72 and CD83. The HSV-IPC expression of CD13, CD33 and Fc epsilon RI were weak but significant, while no CD5, CD11b, CD16, CD64, CD80 or CD86 were detected. Sorted pure HSV-IPC had irregular shaped nuclei, many mitochondria and vesicles, and rugged cell membranes without veils. Sorted HSV-IPC stimulated proliferation of autologous T cells from HSV immune donors. Thus, the HSV-IPC in many respects resemble immature DC, but clearly differ from typical mature DC. However, they may also represent a specialized population of efficient IFN-alpha producing cells.
Human blood mononuclear leukocytes (PBMCs) producing interferon-alpha (IFN-alpha) after stimulation by herpes simplex virus type 1 (HSV) in vitro were identified by a filter immuno-plaque assay. Individual IFN-alpha-producing cells (IPCs) yielded between 0.5 and 2 units IFN-alpha, sufficient to protect cultures of MDBK cells against a viral challenge. Therefore, their frequency could be determined by a limiting dilution assay as well as by an immuno-plaque assay. Similar estimates of between 2 and 55 IPCs per 10(4) PBMCs at the peak of the IFN-alpha response were obtained by the two methods. IPCs were first detected 3 h after stimulation by HSV; their number peaked at 8 h and then declined. IPC frequencies were influenced by the concentrations of HSV and PBMCs during induction, but the quantity of IFN-alpha produced per IPC was relatively constant. The relation between the numbers of IPCs and PBMCs was linear at high PBMC concentrations, whereas at low PBMC concentrations fewer IPCs than expected were detected. The response could be fully restored by adding a combination of filler cells (Namalwa or U937 cells) and conditioned medium from 6-h HSV-induced PBMC cultures. Our results suggest that HSV induces an IFN-alpha response in a relatively rare population of efficient IPCs by complex mechanisms, which may involve cell cooperation and/or production of soluble factors.
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