Interactions among type I and type II interferon, tumor necrosis factor, and beta-estradiol in the regulation of immune response-related gene expressions in systemic lupus erythematosus

Abstract

Introduction: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various clinical manifestations. Several cytokines interact and play pathological roles in SLE, although the etiopathology is still obscure. In the present study we investigated the network of immune response-related molecules expressed in the peripheral blood of SLE patients, and the effects of cytokine interactions on the regulation of these molecules.

Methods: Gene expression profiles of peripheral blood from SLE patients and from healthy women were analyzed using DNA microarray analysis. Differentially expressed genes classified into the immune response category were selected and analyzed using bioinformatics tools. Since interactions among TNF, IFNgamma, beta-estradiol (E2), and IFNalpha may regulate the expression of interferon-inducible (IFI) genes, stimulating and co-stimulating experiments were carried out on peripheral blood mononuclear cells followed by analysis using quantitative RT-PCR.

Results: Thirty-eight downregulated genes and 68 upregulated genes were identified in the functional category of immune response. Overexpressed IFI genes were confirmed in SLE patient peripheral bloods. Using network-based analysis on these genes, several networks including cytokines--such as TNF and IFNgamma--and E2 were constructed. TNF-regulated genes were dominant in these networks, but in vitro TNF stimulation on peripheral blood mononuclear cells showed no differences in the above gene expressions between SLE and healthy individuals. Co-stimulating with IFNalpha and one of TNF, IFNgamma, or E2 revealed that TNF has repressive effects while IFNgamma essentially has synergistic effects on IFI gene expressions in vitro. E2 showed variable effects on IFI gene expressions among three individuals.

Conclusions: TNF may repress the abnormal regulation by IFNalpha in SLE while IFNgamma may have a synergistic effect. Interactions between IFNalpha and one of TNF, IFNgamma, or E2 appear to be involved in the pathogenesis of SLE.

Figure 1

Network-based analysis of downregulated genes in the functional category of immune response. (a) Network 1 and (b) Network 2 constructed by downregulated genes. (c) Network graphical representation. Genes or gene products are represented as individual nodes whose shapes represent the functional class of gene products. The biological relationship between the two nodes is represented as an edge (line). All edges are supported by at least one reference from the literature stored in the Ingenuity Pathways Knowledge Base (IPKB). Genes in colored nodes were found over-represented in the functional category of immune response. Genes in uncolored nodes were not found over-represented but were depicted by the computationally generated networks on the basis of evidence stored in the IPKB indicating a strong biologic relevance to that network.

Figure 2

Network-based analysis of upregulated genes in the functional category of immune response. (a) Network 1, (b) Network 2, (c) Network 3, and (d) Network 4 constructed by upregulated genes.

Figure 3

Effect of TNF stimulation on gene expression in healthy individuals and systemic lupus erythematosus patients. Peripheral blood mononuclear cells (PBMCs) from six systemic lupus erythematosus (SLE) patients and three healthy individuals (HI) were isolated and stimulated for 24 hours in the absence and presence of 20 ng/ml TNF. The relative mRNA expressions (RE) compared between TNF-stimulated and nonstimulated control individuals were measured using quantitative RT-PCR. The RE of seven downregulated genes (highlighted in green) and 12 upregulated genes (highlighted in red) are designated by five colors as shown. See Table 1 for gene identification.

Figure 4

Effect of cytokines or β-estradiol on the expressions of interferon-inducible genes. Peripheral blood mononuclear cells from three healthy donors were cultured with the indicated cytokines for 24 hours. RNA was analyzed by quantitative RT-PCR as described in Materials and methods. Relative expression of the indicated genes – interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), and interferon alpha-inducible protein 27 (IFI27) – compared with their nonstimulated cultures is shown. Each bar represents the mean value of duplicate wells as compared with the nonstimulated control. Downregulated genes were arbitrarily assigned a negative value.