Distinct evolution of TLR-mediated dendritic cell cytokine secretion in patients with limited and diffuse cutaneous systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease and accumulating evidence suggests a role for Toll-like receptor (TLR)-mediated activation of dendritic cells (DCs). OBJECTIVE: To map TLR-mediated cytokine responses of DCs from patients with SSc. METHODS: 45 patients with SSc were included. Patients were stratified as having diffuse cutaneous SSc (dSSc) or limited cutaneous SSc (lSSc) according to the extent of skin involvement, and further divided into those with late (>3 years) or early disease (<2 years). DCs were stimulated with ligands for TLR2, TLR3, TLR4, TLR7/8 or combinations. Plasma samples were collected from patients with SSc (n=167) and measured for interleukin 6 (IL-6), tumour necrosis factor alpha (TNFalpha), IL-12, IL-10 and interferon gamma. RESULTS: Stimulation of DC subsets from patients with early lSSc and dSSc with ligands for TLR2, TLR3 or TLR4 resulted in higher secretion of IL-6 and TNFalpha compared with those having late disease or healthy controls. Remarkably, the production of IL-12 was lower upon stimulation with TLR ligands in most patients with SSc, whereas the secretion of IL-10 was very high in patients with the dSSc phenotype, particularly in those having early dSSc. The combination of various TLR ligands led to reduced cytokine secretion in all patients with SSc. Circulating levels of these cytokines further underscored the presence of differences between various SSc phenotypes. DISCUSSION: The altered TLR-mediated activation of DCs may be responsible for Th2 skewed T-cell activation in SSc that may be orchestrated by fibrogenic T-cell cytokines, such as IL-4 and IL-13. DC targeting could thus offer new avenues for therapeutic intervention.

A four-gene biomarker predicts skin disease in patients with diffuse cutaneous systemic sclerosis.

OBJECTIVE: Improved outcome measures in systemic sclerosis (SSc) are critical to finding active therapeutics for this disease. The modified Rodnan skin thickness score (MRSS) is the current standard for evaluating skin disease in SSc, but it is not commonly used in the clinical setting, in part because it requires specialized training to perform accurately and consistently. The purpose of this study was to investigate whether skin gene expression might serve as a more objective surrogate outcome measure to supplement skin score evaluations. METHODS: Skin RNAs from a group of patients with diffuse cutaneous SSc were studied for expression levels of genes known to be regulated by transforming growth factor beta (TGFbeta) and interferon (IFN). These levels were correlated with the MRSS, using multiple regression analyses to obtain best-fit models. RESULTS: Skin expression of the TGFbeta-regulated genes cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP-1) correlated moderately well with the MRSS, but the addition of other TGFbeta-regulated genes failed to significantly improve best-fit models. IFN-regulated genes were also found to correlate with the MRSS, and the addition of interferon-inducible 44 (IFI44) and sialoadhesin (Siglec-1) to COMP and TSP-1 in multiple regression analyses significantly improved best-fit models, achieving an R(2) value of 0.89. These results were validated using an independent group of skin biopsy samples. Longitudinal scores using this 4-gene biomarker indicated that it detects change over time that corresponds to changes in the MRSS. CONCLUSION: We describe a 4-gene predictor of the MRSS and validate its performance. This objective measure of skin disease could provide a strong surrogate outcome measure for patient care and for clinical trials.

Enhanced interleukin-10 production by dendritic cells upon stimulation with Toll-like receptor 4 agonists in systemic sclerosis that is possibly implicated in CCL18 secretion.

BACKGROUND: It has been suggested that the T-cell attracting and profibrotic chemokine CCL18 might play a role in the pathogenesis of systemic sclerosis (SSc). However, it is unclear what underlies the higher CCL18 levels in SSc. The aim of our study was to determine whether Toll-like receptor (TLR)-mediated stimulation of monocytes and dendritic cells (DCs) contributes to the higher levels of CCL18 in SSc. METHODS: CCL18 levels were measured in 40 patients with SSc, primary Raynaud's phenomenon (RP) and healthy controls. The presence of TLR4 agonists in the circulation of SSc patients was investigated using TLR4 transgenic Chinese hamster ovary (CHO) cells. CCL18 and interleukin (IL)-10 secretion by monocytes/macrophages and monocyte-derived DCs (moDCs) was measured in the supernatant. The indirect effect of lipopolysaccharide (LPS)-stimulated moDCs on CCL18 secretion by monocytes/macrophages was investigated using a transwell system. RESULTS: CCL18 levels were significantly elevated in SSc patients compared to patients with RP and healthy controls. SSc sera strongly induced CD25 expression on CHO cells genetically modified to express TLR4 but not on those expressing CD14 only. By contrast, serum from systemic lupus erythematosus (SLE) patients or healthy individuals did not have an effect. Neither monocytes/macrophages nor moDCs from SSc patients secreted higher levels of CCL18 compared to healthy controls. However, moDCs matured with the TLR4 ligand LPS from patients with SSc did secrete significantly higher amounts of IL-10 compared to those from healthy counterparts, which were IL-10 dependent. CONCLUSIONS: Our results suggest that elevated CCL18 levels in SSc are not caused by an intrinsically enhanced CCL18 secretion by monocytes/macrophages but are, at least partly, orchestrated by an enhanced IL-10 secretion by TLR4-stimulated DCs. These observations suggest a role for TLR4 ligands and DCs in the pathogenesis of SSc, a topic that warrants further investigation.

Cartilage oligomeric matrix protein expression in systemic sclerosis reveals heterogeneity of dermal fibroblast responses to transforming growth factor beta.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) accumulates in systemic sclerosis (SSc) skin and is upregulated by transforming growth factor (TGF)beta. To further characterise the response to TGFbeta in SSc, we investigated TGFbeta1 and COMP expression and myofibroblast staining in SSc skin. METHODS: Skin biopsies from patients with diffuse cutaneous SSc (dSSc), limited cutaneous SSc (lSSc) and healthy controls were evaluated for COMP mRNA expression using real-time PCR. COMP, alpha-smooth muscle actin (SMA) and TGFbeta were assessed in skin sections and in cultured fibroblasts by immunohistochemistry. Clinical disease status was assessed by the modified Rodnan skin score (mRSS). RESULTS: Myofibroblasts expressing SMA and COMP were found coexpressed in many cells in dSSc dermis, but each also stained distinct cells in the dermis. Cultured SSc dermal fibroblasts also showed heterogeneity for COMP and SMA expression, with cells expressing SMA, COMP, both or neither. TGFbeta treatment increased COMP and SMA-expressing cells. COMP mRNA expression in lesional skin from patients with dSSc correlated with the mRSS and TGFbeta1 staining. CONCLUSION: These findings suggest that TGFbeta upregulation of COMP and/or SMA expression in subpopulations of fibroblasts contributes to different pathways of fibrosis and that multiple TGFbeta regulated genes may serve as biomarkers for the degree of SSc skin involvement.

Assessment of strategies for identifying diagnosed cases of systemic lupus erythematosus through self-report.

The objective of this work was to assess the optimal way to identify potential systemic lupus erythematosus (SLE) cases in large epidemiologic studies through self-reported information about diagnosis of SLE, symptoms and medications, and to investigate the utility of a criteria checklist sent directly to participants' physicians. We used data collected in 1997 from 53322 participants in a study of African-American women, the Black Women's Health Study, including a lupus screening questionnaire (LSQ) and questions about SLE diagnosis and medications. We confirmed self-reported SLE through medical records and criteria checklists sent to participants' physicians. Among those for whom we received medical records and/or criteria checklists, we compared the predictive value and proportion of missed cases of several algorithms using combinations of self-reported SLE diagnosis, LSQ score and medication use to self-reported SLE diagnosis alone. We obtained a physician checklist or medical chart for 251 individuals who reported SLE, of whom 212 (84%) fulfilled ACR criteria for definite or probable SLE, or had clinical lupus (SLE diagnosis recorded in medical charts plus appropriate medication use). The use of LSQ score or medication use in addition to self-report of SLE tended to decrease the false positive rate but also to reduce the proportion of true cases identified. Checklists of ACR criteria completed by subjects' physicians documented more criteria than medical records. In conclusion, among participants who consented to medical record review, SLE prediction algorithms using questions about lupus symptoms and medications offered slightly higher predictive value for detecting cases than self-reported diagnosis alone, but at the cost of case detection. SLE case confirmation strategies can be complemented by the use of criteria checklists sent directly to participants' physicians.

Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult-specific and that codes for a microtubule binding domain. Recently, mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we establish the endogenous expression patterns of exon 10 in human tissue; by reconstituting naturally occurring FTDP mutants in the homologous context of exon 10, we show that the cis determinants of exon 10 splicing regulation include an exonic silencer within the exon, its 5' splice site, and the relative affinities of its flanking exons to it. By cotransfections in vivo, we demonstrate that several splicing regulators affect the ratio of tau isoforms by inhibiting exon 10 inclusion.

SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation.

CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO.

Integrin engagement regulates proliferation and collagenase expression of rheumatoid synovial fibroblasts.

Growth of and metalloproteinase production by fibroblast-like synoviocytes (FLSs) in patients with rheumatoid arthritis (RA) contribute to cartilage and bone destruction associated with development of the expanding inflammatory tissue referred to as pannus. Increased levels of extracellular matrix (ECM) proteins in the pannus suggest that intracellular signals generated through integrin receptors might control these processes. We developed a cell culture system permitting accurate assessment of the effect of cell adhesion to various ECM proteins on FLS phenotype. We show that FLS proliferation to platelet-derived growth factor requires a second signal provided by adhesion to an ECM protein. Fibronectin, vitronectin, collagen, or laminin could provide the second signal and was similarly required for the proliferation of FLSs from RA or osteoarthritis patients. Adhesion to fibronectin, collagen, or Arg-Gly-Asp peptide down-regulated collagenase expression. Primarily alphav integrin receptors mediated this down-regulation upon adhesion to fibronectin. Loss of cell adhesion and TNF-alpha stimulation synergistically increased collagenase expression. Increased collagenase expression upon nonadherence was mimicked by treatment with cytochalasin B, suggesting that the loss of cytoskeletal structure associated with a change in cell shape mediates increased collagenase in nonadherent cells. Thus, although increased fibronectin in the lining layer in RA might be expected to inhibit collagenase expression, the change in cell shape associated with this multilayer structure might actually lead to increased collagenase expression.

Transforming growth factor-beta and platelet derived growth factor regulation of fibrillar fibronectin matrix formation by synovial fibroblasts.

OBJECTIVE: To investigate factors regulating fibronectin fibrillar matrix formation by synovial fibroblasts. METHODS: Basal and cytokine stimulated extracellular matrix (ECM) fibronectin produced by synovial fibroblasts was identified by immunofluorescence and Western blot. Alternative mRNA splicing of fibronectin was studied by reverse transcription polymerase chain reaction. The integrin receptor responsible for supporting fibronectin fibrillar matrix was identified by blocking antibodies and receptor levels studied by Western blot. RESULTS: Transforming growth factor-beta (TGF-beta) or platelet derived growth factor (PDGF), but not interleukin 1 or exogenous fibronectin, induced ECM fibronectin. ECM fibronectin was blocked by the addition of antibody to the alpha5beta1 integrin. Cytokines did not significantly change alternative mRNA splicing of fibronectin or levels of alpha5beta1 integrin expression. CONCLUSION: Synovial cell production of a fibrillar fibronectin matrix is induced by growth factors, including TGF-beta and PDGF. This induction is mediated by the alpha5beta1 integrin. Since fibrillar fibronectin formation was not strongly dependent on increased fibronectin or alpha5beta1 integrin levels, this effect may be mediated by growth factor induced changes in receptor affinity.

The mammalian homolog of suppressor-of-white-apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS.

We have previously described human (HsSWAP) and mouse (MmSWAP) homologs to the Drosophila alternative splicing regulator suppressor-of-white-apricot (su(wa) or DmSWAP). DmSWAP was formally defined as an alternative splicing regulator by studies showing that it autoregulates splicing of its own pre-mRNA. We report here that mammalian SWAP regulates its own splicing, and also the splicing of fibronectin and CD45. Using an in vivo system of cell transfection, mammalian SWAP regulated 5' splice site selection in splicing of its own second intron. SWAP enhanced splicing to the distal 5' splice site, whereas the SR protein ASF/SF2 enhanced splicing to the proximal site. SWAP also regulated alternative splicing of the fibronectin IIICS region by promoting exclusion of the entire IIICS region. In contrast, ASF/SF2 stimulated inclusion of the entire IIICS region. Finally, SWAP regulated splicing of CD45 exon 4, promoting exclusion of this exon, an effect also seen with ASF/SF2. Experiments using SWAP deletion mutants showed that splicing regulation of the fibronectin IIICS region and CD45 exon 4 requires a region including a carboxyl-terminal arginine/serine (R/S)-rich motif. Since R/S motifs of various splicing proteins have been shown to interact with each other, these results suggest that the R/S motif in SWAP may regulate splicing, at least in part, through interactions with other R/S containing splicing factors.

Synovial fibroblast-like cell transfection with the SV40 large T antigen induces a transformed phenotype and permits transient tumor formation in immunodeficient mice.

OBJECTIVE: To understand the intracellular signals leading to transformed-like growth of synovial fibroblast-like cells from patients with rheumatoid arthritis (RA). METHODS: Cell lines stably transfected with one or both of 2 complementary oncogenes, the SV40 large T antigen and the ras oncogene, were studied for phenotypic changes. RESULTS: Synovial fibroblast-like cells stably transfected with the SV40 large T antigen, but not the ras oncogene, showed high levels of growth factor independent proliferation, grew under anchorage independent conditions, expressed cathepsin L mRNA, and formed transient tumors in immunodeficient mice. Synovial fibroblast-like cells stably transfected with both oncogenes appeared phenotypically similar to synovial fibroblast-like cells transfected with the large T antigen alone. CONCLUSION: The SV40 large T antigen confers a phenotype on synovial fibroblast-like cells similar to that stimulated by growth factors, suggesting that it stimulates the same intracellular signalling pathway leading to cytokine induced, transformed synovial fibroblast-like cell growth. When injected into immunodeficient mice these transfected cells formed tumors characterized by rapid, transient growth, central necrosis, and neutrophil infiltration.

The state of differentiation of HT-29 colon carcinoma cells alters the secretion of cathepsin D and of plasminogen activator.

We have studied the cellular content and the extracellular release of cathepsins B and D, and of plasminogen activator, in 2 different tumor cell populations before confluence and after late confluence: the HT-29 colon carcinoma cell line, which contains primarily undifferentiated cells, and a subpopulation derived from this cell line, which contains cells committed to differentiation into mucus-secreting goblet cells after confluence. In both populations, cellular cathepsin-B activity increased after confluence, and latent cathepsin B was found in all culture media. In the parental cell line, cellular cathepsin D activity decreased after confluence; however, cathepsin D was secreted at high levels into the extracellular medium. In contrast, in the subpopulation of cells committed to differentiation, cellular cathepsin D activity increased after confluence, and cathepsin D was not secreted into the extracellular medium, but was immunolocalized in the apical brush border of the differentiated cells. Plasminogen activator of urokinase type was identified by immunocytochemistry. Both subconfluent cell populations, and the post-confluent undifferentiated cell population, produced plasminogen activator activity at similar levels. In contrast, in the differentiated postconfluent cells, the production of plasminogen activator activity was markedly lower. Our data show that the differentiation of HT-29 colon carcinoma cells into mucus-secreting cells impairs the secretion of plasminogen activator and cathepsin D, but does not affect cathepsin B.

Conservation of regulated alternative splicing and identification of functional domains in vertebrate homologs to the Drosophila splicing regulator, suppressor-of-white-apricot.

Although several splicing regulatory proteins have been identified in Drosophila through characterization of various genetic mutations, including sex-lethal, transformer, transformer-2, suppressor-of-white-apricot (su(wa)), and possibly suppressor-of-sable, none of these have been identified in vertebrates. We describe the cloning and characterization of human (HsSWAP) and mouse (MmSWAP) homologs of the su(wa) gene. Comparison of the Drosophila and mammalian proteins reveals five highly homologous regions, including an arginine/serine-rich domain and two repeated modules that are homologous to regions in the constitutive splicing factor, SPP91/PRP21. These modules thus define a new motif likely important in the regulatory and constitutive splicing functions of these proteins. The Drosophila su(wa) gene autoregulates its expression by control of splicing of its first two introns. Comparison of mammalian and Drosophila SWAP mRNAs revealed that the splice junctions of these regulated introns are precisely conserved, showing definitively that these genes are ancestrally related. Moreover, mammalian SWAP mRNAs are also alternatively spliced at the same splice sites, showing that mammalian SWAP expression is regulated (presumably autogenously) by control of splicing of these two introns. These several structural features therefore strongly suggest that the mammalian SWAP gene functions as a vertebrate alternative splicing regulator.

Regulation of the transforming growth factor-beta 1 and -beta 3 promoters by transcription factor Sp1.

The promoter regions of the genes encoding the three mammalian transforming growth factors-beta (TGF-beta 1, -beta 2, and -beta 3) show little similarity in sequence, suggesting diverse transcriptional control. As a step towards understanding transcriptional regulation of the individual TGF-beta genes we tested each of the three TGF-beta promoter regions (pTGF-beta) for stimulation by the transcription factor Sp1, given that several possible Sp1-binding sites were identified by sequence analysis in pTGF-beta 1 and pTGF-beta 3. A Drosophila melanogaster cell culture system was employed to examine expression levels of pTGF-beta::cat constructs coexpressed with an Sp1 expression plasmid in a cell background devoid of any Sp1 homolog. While both pTGF-beta 1 and pTGF-beta 3 were strongly stimulated by Sp1, pTGF-beta 2 was completely unaffected. Promoter fragments of the TGF-beta 1 and TGF-beta 3 genes, but not TGF-beta 2 were able to compete for binding of Sp1 to DNA oligomers containing consensus Sp1-binding sites. Moreover, specific binding to pTGF-beta 1 and pTGF-beta 3 fragments was seen using pure Sp1 or nuclear protein extracts. Thus, TGF-beta 1 and TGF-beta 3 (but not TGF-beta 2) are regulated by the transcription factor Sp1, indicating differential transcriptional regulation of genes whose protein products are functionally very similar.

Stimulation of the secretion of latent cysteine proteinase activity by tumor necrosis factor alpha and interleukin-1.

OBJECTIVE: Cultured synovial fibroblast-like cells from 3 patients with rheumatoid arthritis (RA) and 3 patients with osteoarthritis (OA) were evaluated for their potential to secrete cysteine proteinases spontaneously and after stimulation by tumor necrosis factor alpha (TNF alpha) or interleukin-1 (IL-1). METHODS: Culture media and cell lysates were analyzed before and after high performance liquid chromatography (HPLC) using the enzymatic substrate, Z-Phe-Arg-AMC, and by immunoblotting with anti-cathepsin B antiserum. Immunolocalization of cathepsin B was studied on cell monolayers. RESULTS: Latent cysteine proteinase activity was found to be secreted spontaneously by cultured synovial fibroblast-like cells. This activity was increased after treatment with either TNF alpha or IL-1. Stimulated protease activity was eluted by HPLC at a peak coincident with that of purified cathepsin B. By immunoblot, cell supernatants contained a 43-kd form of cathepsin B, while cell lysates contained a 30-kd form, consistent, respectively, with cathepsin B before and after cleavage of its propeptide. An intracellular increase in cathepsin B after treatment with TNF alpha was also seen with immunohistochemical studies. CONCLUSION: TNF alpha (in the 6 cases studied) and IL-1 (in 4 cases) stimulated the secretion of a latent cysteine proteinase activity from synovial fibroblast-like cells, which appears to represent primarily cathepsin B.

Antibodies in rheumatoid synovial fluids bind to a restricted series of protein antigens in rheumatoid synovial tissue.

OBJECTIVE: By searching the synovial fluid of patients with rheumatoid arthritis (RA) for antibodies that react to protein antigens in synovial tissue, we sought to identify putative antigens present in RA synovial tissue that might drive the pathologic immune response believed to be responsible for the joint inflammation. METHODS: Synovial tissue was homogenized in sodium dodecyl sulfate polyacrylamide gel buffer, electrophoresed, and analyzed by immunoblotting. RESULTS: Antibodies from synovial fluids of patients with RA bound to several proteins in rheumatoid synovial tissues, including a series of low (27.5-, 29-, and 30-kd), middle (43- and 53-kd), and high (140-, 164-, and 182-kd) molecular weight proteins. Most of these antigens were also detected in normal synovial tissue, and the high molecular weight proteins were also present in normal dermal, muscle, and liver tissues. The low and middle molecular weight proteins were detected in some, but not all, of the other normal tissues and in Jurkat cell lysates. Antibodies to the low and high series of proteins were present in all rheumatoid synovial fluids tested, but were generally absent from synovial fluids from patients with other arthritic diseases. CONCLUSION: These results show that antibodies in synovial fluids consistently react to several proteins in RA and normal synovial tissues. These antigens are possibly the same antigens provoking the T cell response in RA; therefore, understanding the mechanism of the immune response against these proteins will likely lead to important insight into the etiology of RA.

Sequence specific protein binding to and activation of the TGF-beta 3 promoter through a repeated TCCC motif.

We have previously characterized the TGF-beta 3 promoter and shown that the activity of this promoter is highly variable in different cell types. Although the promoter contains a proximal cAMP responsive element, which is critical to basal and forskolin-induced promoter activity, this element is not responsible for the variable, cell-specific regulation of the promoter. In this paper, we identify a 25 base pair sequence in the proximal region of the TGF-beta 3 promoter that binds a novel DNA-binding protein. This region includes the sequence T-CCCTCCCTCCC, (3 x TCCC), and mutation of these T-CCC repeats inhibits protein binding. Further, we show that in the cell line A375, which we have previously shown expresses high levels of TGF-beta 3 mRNA, this region is responsible for mediating high level TGF-beta 3 promoter activity. Immediately 3' to the 3 x TCCC sequence is a consensus AP-2 binding site, however, we show that this region does not bind AP-2, and AP-2 does not transactivate the TGF-beta 3 promoter. Therefore, we provide strong evidence that high level expression of TGF-beta 3 in A375 cells results from transactivation of the TGF-beta 3 promoter by a protein that binds to a repeated TCCC motif in the promoter and suggest that this DNA-binding protein likely also regulates aspects of developmental and tissue-specific expression of this cytokine.