Interferon and Granulopoiesis Signatures in Systemic Lupus Erythematosus Blood

Systemic lupus erythematosus (SLE) * affects multiple organs, including the skin, vessels, kidneys, and central nervous system ( 1 , 2 ). SLE etiopathogenesis has challenged investigators for many years. It is believed that the environment, e.g., viral infections, acts in the context of a wide array of susceptibility genes. Human SLE appears linked to at least 6 gene loci and numerous mouse genetic mutants show SLE-like syndromes ( 3 ). This disease, where tolerance to self-components is broken in a systemic fashion, is currently viewed as a dysregulation of T–B cell interactions ( 4 – 7 ). SLE patients paradoxically display polyclonal hypergammaglobulinemia concomitantly with B and T lymphopenia ( 8 , 9 ). Indeed, human SLE is characterized by high titers of autoantibodies of a wide range of specificities, including nuclear components, DNA, and nucleosomes ( 10 – 12 ). Vasculitis and kidney failure are thought to be immune complex-mediated while other symptoms such as thrombocytopenia are viewed as direct antibody-mediated depletion. Yet, these alterations represent end-points of a dysregulated immune system. Therefore, understanding the early stages of the disease might lead to the development of better therapies, which currently are symptomatic and based on nonspecific immunosuppression i.e., glucocorticoids and chemotherapy. Dendritic cells (DCs; reference 13 ) are being recognized as critical in the maintenance of peripheral tolerance ( 14 ). Therefore, we turned our attention to their potential alteration in pediatric SLE, a disease of high morbidity/mortality. Accordingly, we demonstrated that a fraction of patients display in their blood stream monocytes with properties of DCs, namely the ability to induce allogeneic naive T cells to proliferate (MLR; reference 15 ). Furthermore, the serum of some patients induced the differentiation of normal monocytes into DCs, an effect mediated by IFN-α. This led us to consider that SLE may be driven by unabated IFN production that activates monocytes into mature DCs able to capture dying cells and present their antigens to autoreactive T/B cells. Yet, only a fraction of patients display circulating IFN-α ( 16 ), thereby raising the question of whether this reflects disease heterogeneity. Because IFN-α may represent a major therapeutic target in SLE, as TNF does in arthritis ( 17 ), we approached this question by analyzing the genes expressed by patient leukocytes using oligonucleotide microarray technology ( 18 – 20 ). Here we demonstrate that blood mononuclear cells from active SLE patients overexpress IFN-regulated and granulopoiesis-specific genes. The IFN signature is extinguished by treatment with high dose intravenous steroids, further pointing to this cytokine as a specific target for therapeutic intervention.

A fraction of patients cells was retained for flow cytometry analysis including the determination of T cells, B cells, plasmablasts (CD20−CD19 low CD38 high), monocytes as described earlier ( 9 ) and DCs (DC kit; Becton Dickinson). Leukocytes from healthy adults were cultured in RPMI enriched with 5% fetal calf serum for 6 h with or without 1,000 U/ml Interferon a2b (Intron A; Schering-Plough).

30 pediatric SLE patients, 18 females and 12 males, were enrolled in the study Dec 2001–June 2002. Of these, 13 were Hispanic, 8 African-American, 6 Asian, and 3 Caucasian. This distribution reflects the overall ethnicity of our SLE population in North Texas. The detailed clinical information is provided as online supplemental Table S1.

An absolute expression analysis was performed using Affymetrix MAS 5.0, and the data from 12,626 genes was imported into GeneSpring software (Silicon Genetics) for further analyses. Differentially expressed genes were selected as described in Results.

Gene Expression in Blood Leukocytes. To identify gene expression signatures unique to SLE, we analyzed Ficoll-separated blood mononuclear cells (PBMCs) from three pediatric cohorts: 9 healthy children (age 13 ± 4 yr), 30 SLE patients (age 13 ± 3 yr), and 12 patients with juvenile chronic arthritis (JCA; age 10 ± 3 yr). SLE patients belonged to four ethnicities and included 12 males and 18 females. One patient had been in complete remission (SLE Disease Activity Index [SLEDAI] = 0) for more than 2 yr, 11 patients had minimal disease activity (SLEDAI = 2–4), 10 patients had intermediate disease activity (SLEDAI = 6–10) and 8 patients had high disease activity (SLEDAI = 11–20). The clinical features are provided as supplementary data (Table S1). Of the 12 patients with JCA, 4 had pauci-articular, 5 poly-articular, and 3 the systemic form of the disease. PBMC cRNA was hybridized to U95AV2 Affymetrix oligonucleotide microarrays containing 12,561 human genes and the data were analyzed with the GeneSpring software. Up to 5,000 genes were expressed, with 4,600 being common to all children. The pattern of expression was remarkably stable among individuals. SLE patients and healthy donors were analyzed using a parametric statistical group comparison incorporating the global error method. Using the most stringent multiple comparison correction for controlling Type I error (Bonferroni correction), 15 genes were found differentially expressed (P < 0.1) between these two populations and highly up-regulated in SLE patients. Strikingly, 14 of 15 genes ( Table I ) represent either well-known or newly identified targets of interferon. The single non-IFN–regulated gene was defensin, a major product of immature neutrophils. Applying a more liberal correction to the pairwise tests (Benjamini and Hochberg correction) yielded 18 additional genes, 12 of which are known to be IFN-regulated and 4 neutrophil-specific ( Table I ). Use of the more liberal multiple comparison corrective technique yields an expanded list of genes differentially expressed (P < 0.05) between these two groups to 374 ( Fig. 1 ) . Among these, 210 genes were up-regulated and 141 genes were down-regulated in a majority of patients. Table I. Genes Significantly Up-regulated in SLE Patient's Blood Mononuclear Cells GenBank accession no. Description Family Function P value Bonferroni P value Benjamini and Hochberg M87434 71 kD 2′5′ OIAS IFN Antiviral <0.00001 <0.0001 AB000115 GS3686 IFN Unknown <0.0001 <0.0001 D28195 Hep C p44 IFN Unknown 0.0001 <0.0001 X57352 1-8U IFN Unknown <0.0002 <0.0001 M33882 MX1 IFN GTP-ase antiviral <0.0003 <0.0001 M30818 MX2 IFN GTP-ase antiviral <0.002 <0.0003 U66711 RIGE/TSA1 IFN Signal transduction <0.01 <0.001 AB006746 Phospholipid scramblase IFN Transbilayer migration of phospholipids <0.03 <0.003 L12691 DEFA3 Neut Antibacterial <0.03 <0.003 X04371 2′5′ OIAS E18 isoform IFN Antiviral degradation of RNA <0.03 <0.003 AL047596 EST Hute 1 IFN Unknown <0.05 <0.005 U53831 IRF 7b IFN Transcription activator <0.05 <0.005 M97935 ISGF-3 IFN Transcription factor 0.06 <0.005 AL022318 Phorbolin 1 like IFN Unknown mRNA editing? 0.06 <0.005 U72882 IF p35 IFN Unknown 0.07 <0.005 L13210 MAC-2-BP IFN Host defense cell adhesion <0.02 X99699 XIAP associated factor 1 IFN Proapoptotic <0.02 M13755 ISG-15 IFN Unknown <0.03 X69910 p63 transmembrane protein Unknown Membrane trafficking <0.03 X54486 C1 inhibitor IFN C1 esterase inhibition <0.03 X55988 Eosinophil derived neurotoxin Neut Ribonuclease <0.04 L09708 Complement component 2 IFN Complement cascade <0.05 AF016903 Agrin IFN Aggregation of signaling proteins at the neurological and immunological synapsis <0.05 X57522 TAP1 IFN Antigen presentation <0.05 AF026939 Cig 49 IFN Unknown <0.05 AI126134 EST similar to calgranulin Neut Calcium binding protein proinflammatory <0.05 AJ225089 TRIP 14 OIAS IFN Antiviral degradation of RNA <0.05 U37518 TRAIL IFN Apoptosis <0.05 M84562 Formyl peptide receptor-like Neut Neutrophil activation and chemotaxis <0.05 AL036554 DEFA1 Neut Antibacterial 0.05 Z38026 FALL-39 Neut Antibacterial 0.06 AB025254 Sim to Dros. Tudor IFN Unknown 0.06 M24594 IFI-56 IFN Unknown 0.06 Figure 1. SLE signature. Hierarchical clustering of gene expression data by blood leukocytes of 9 healthy children, 30 with SLE, and 12 with juvenile chronic arthritis including 3 systemic arthritis. The SLE patients have been ranked according to their SLEDAI at time of blood draw. Each row represents a separate gene and each column a separate patient. 374 transcript sequences have been selected which were differentially expressed in SLE by comparison to healthy patients. The normalized expression index for each transcript sequence (rows) in each sample (columns) is indicated by a color code. Red, yellow, and blue squares indicate that expression of the gene is greater than, equal to o

Numerous lymphocyte-specific transcripts were decreased in SLE PBMCs, an expected finding in line with the lymphopenia that characterizes human SLE. In particular, the pan-T cell genes TCRα/β, p56 lck , CD3γ chain, and SAP were down-regulated in many patients ( Fig. 3 A). Pan-B cell (CD20, CD79a/b, and CD22) and naive B cell markers (IgD) were dowregulated in 14 patients. Figure 3. Lymphoid signature: T and B lymphopenia with hypergammaglobulinemia. (A) T cell genes, and the Ig genes. Median expression and the number of patients who display more than twofold increase (red) or decrease (blue) in gene expression. (B) Flow cytometry analysis of purified B cells showing the high frequency of CD19 + CD38 high plasmablasts. (C) IgG transcript levels correlate with plasmablast numbers. On the contrary, >2 fold up-regulated transcription of IgG and IgA was found in 14 and 7 patients, respectively, and correlated with their number of circulating plasma blasts/cells ( Fig. 3, B and C ). There was a strong correlation between increased IgG transcription and gender, as 2/12 males and 12/18 females up-regulated this molecule. Additionally, down-regulated (<2 fold) IgG transcripts were found in 5/12 males but in only 1/18 females (P = 0.009, Chi square with exact P value). CD161, a marker of NK/NKT cells, was found to be the most significantly decreased (19/30 patients) lineage marker. Two genes specific for activated DCs, DC-LAMP and CD83, were up-regulated in 16 and 6 patients, respectively. Furthermore, TAP1, a critical molecule for MHC class I antigen presentation, was up-regulated in 6 patients ( Fig. 2 A) as well as in the in vitro IFN-treated PBMCs, thus extending the list of IFN-regulated genes. This pattern of DC activation molecules is consistent with our previous report demonstrating unabated DCs induction by SLE serum ( 15 ).

Out of 33 SLE-specific genes selected with the Benjamini and Hochberg multiple comparison correction, we could identify a set of 10 whose expression correlated with disease activity according to the SLEDAI ( Table III , Fig. 6 ) . Of these, 8 genes were IFN-regulated and 2 were neutrophil-related. Expression of every one of these genes correlated better with disease activity than serum levels of anti–double-stranded DNA antibodies, a hallmark of SLE ( Fig. 6 ). The most significant correlation (P < 0.001, r = 0.55) was found with the gene encoding F2RPA, which mediates the chemotactic activities of defensins ( 29 ) and is mainly transcribed within segmented and polymorphonuclear cells ( 26 ; Fig. 6 ). Longitudinal follow-up of these gene expression levels in our patients will allow us to determine their significance in disease flare prediction. This is of particular importance, as there are no parameters other than clinical symptoms to predict SLE flares. Table III. Correlation of Gene Expression with Disease Activity Measured by SLEDAI Gene Family r P value F2RPA Neutr 0.55 0.0014 ISG15 IFN 0.52 0.003 Cig49 IFN 0.50 0.0048 HepC microtubular Agg IFN 0.49 0.0059 TRIP 14 IFN 0.48 0.0069 MX1 IFN 0.43 0.01 Phospholipid scramblase 1 IFN 0.41 0.02 XIAPAF 1 IFN 0.41 0.02 DEFA3 Neutr 0.39 0.03 RIGE/TSA 1 IFN 0.38 0.03 r , Pearson correlation coefficient. Figure 6. Correlation of gene expression and disease activity as measured by SLEDAI. Two IFN-induced genes (Cig49 and phospholipid scramblase 1) and a granulocyte-related gene (F2RPA) correlate significantly with SLEDAI. These particular genes are selected from those in Table III to illustrate different families. By comparison, there is no correlation of serum levels of anti-double stranded DNA antibodies with disease activity.

This study demonstrates that the majority (29/30) of children suffering from SLE display a signature of IFN exposure in their blood cells, therefore reinforcing the role of IFN-α in the pathogenesis of this disease ( 15 , 31 , 32 ). Thus, in spite of being clinically heterogeneous, all active SLE patients regardless of age, gender, and ethnicity have a signature of IFN exposure. Importantly, many of the IFN-α targets represent molecules that have long been associated to SLE pathogenesis. For example, the IFN up-regulated Ro-52 is one of the hallmark antigens in this disease, and autoantibodies against this molecule are routinely measured in SLE patients ( 1 , 2 ). The up-regulation of IFN-induced phospholipid scramblase, TRAIL and XIAPAF1 provides a link to the current association of SLE with dysregulation of apoptosis and/or handling of apoptotic cells ( 33 ). Increased expression of genes coding for TAP1 and DC-LAMP may be relevant for autoantigen presentation. Our studies also show that high dose IV steroid treatment, one of the most effective treatments of SLE flares, efficiently down-regulates the IFN-α signature in SLE leukocytes. This reinforces the importance of targeting IFN to treat this disease, especially as most available therapies, especially glucocorticoids, carry severe morbidity and dramatic life-long side effects, particularly in children. Microarray analysis appears to provide a more faithful indication of exposure of blood cells to IFN than the measurement of IFN in SLE patient's serum. In our previous study, we detected increased levels of IFN-α by ELISA in 10/20 patients ( 15 ). Our current microarray analysis revealed that 29/30 patients had evidence of exposure to this cytokine. Several factors may contribute to the lower sensitivity of the IFN serum assay(s). SLE patients, for example, have been shown to display anti-IFN antibodies in their serum ( 34 ) that may interfere with ELISA measurements. Additionally, the antibody used for ELISA detection may not react with all the IFN species that may circulate in the blood. Finally, our lack of detection of significant IFN-α transcripts in the SLE patient's PBMCs and the reported presence of plasmacytoid DCs in the skin lesions of SLE patients support that this cytokine may be mainly produced in the patient's tissues ( 35 , 36 ). In addition to the IFN signature, we observed a significant up-regulation of granulocyte-specific transcripts within SLE-PBMC RNA. This “granulocyte signature” was traced down to the presence of highly granular cells that copurified with mononuclear cells during density gradient centrifugation. Low density neutrophils have been previously described in the blood of patients with SLE ( 37 ). The highly granular cells that we observe in our patients seem to correspond, however, to granulocytes at different stages of maturation, from myelocytes to polymorphonuclear cells. Giemsa and MPO stainings of PBMCs and CD14-negative granular cells, together with the granulocyte-specific gene transcription pattern that we obtained, support indeed the predominance of immature cells ( 27 ). Low density, mature granulocytes may be contributing, however, to the disease pathogenesis, as the transcription levels of F2RPA, a mature neutrophil transcript, displayed the best correlation with SLE disease activity as measured by the SLEDAI. Stress and demargination are a well known cause of neutrophilia, but they usually yield neutrophils with characteristics of polymorphonuclear cells ( 38 ). Demargination is also associated with steroid treatment. The cells that we describe, however, preferentially transcribe genes characteristic of immature bone marrow precursors. Furthermore, these cells are present not only regardless of steroid treatment, but we find them at the highest levels in new, untreated patients ( Table II ). Our in vitro experiments support that mononuclear cells, i.e., monocytes and lymphocytes, are the main cells responsible for the IFN-α signature. In fact, PBMCs from healthy donors, which do not display the low density granulocyte population, up-regulate the same set of genes upon incubation with IFN for 6 h. Additionally, the IFN signature is evident in five patients who lack the highly granular population and therefore the neutrophil-specific genes ( Table II ). Further experiments to assess the contribution of individual cell population to the SLE blood signatures are currently underway. Hypergammaglobulinemia and immune complex formation represent two classical features of SLE. Pediatric SLE patients, however, display reduced numbers of blood mature lymphocytes and a concomitant expansion of plasma cell precursors ( 9 ). Our current studies confirm these alterations, as we found down-regulation of pan-lymphocyte markers. Additionally, up to half of our patients displayed a blood plasma cell signature with increased transcription of immunoglobulin genes. Interestingly, there was a statistically significant correlatio