Search

Competing Interests: The authors declare no conflict of interest.

Cytokine-primed neutrophils can undergo a nonapoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and NADPH oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF-primed human neutrophils. Specifically, we observed that several inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α) were able to block CD44-mediated reactive oxygen species production and cell death, but not FAS receptor-mediated apoptosis. To understand how FAP-α is involved in this nonapoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 MAPK, PI3K, and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL, and p38 MAPK activation but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis underwent a GM-CSF-independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3-MLKL pathway activates NADPH oxidase but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated nonapoptotic neutrophil death, leading to tissue damage., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Although T H 1, T H 2, and T H 17 cells are well-defined T H cell lineages in humans, it remains debated whether IL-9-producing T H cells represent a bona fide "T H 9" lineage. Our understanding of the cellular characteristics and functions of IL-9-producing T H cells in humans is still nascent. Here, we report that human IL-9-producing T H cells express the chemokine receptors CCR4 and CCR8, produce high levels of IL-5 and IL-13, and express T H 2 lineage-associated transcription factors. In these cells, IL-9 production is activation dependent, transient, and accompanied by down-regulation of T H 2 cytokines, leading to an apparent "T H 9" phenotype. IL-9 + T H 2 cells can be distinguished from "conventional" T H 2 cells based on their expression of the transcription factor PPAR-γ. Accordingly, PPAR-γ is induced in naïve T H cells by priming with IL-4 and TGF-β ("T H 9" priming) and is required for IL-9 production. In line with their identity as early activated T H 2 cells, IL-9 + T H 2 cells are found in acute allergic skin inflammation in humans. We propose that IL-9-producing T H cells are a phenotypically and functionally distinct subpopulation of T H 2 cells that depend on PPAR-γ for full effector functions., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objective: Tocilizumab is effective in inducing and maintaining remission of GCA. Despite clinical and serological control of disease, magnetic resonance angiography may show persistence of inflammatory signals of unknown significance in arterial walls. Thus, there is an unmet need for tools to detect subclinical disease activity., Methods: Immune-inflammatory markers were measured in prospectively collected sera of the first randomized, double-blind, placebo-controlled trial investigating the use of tocilizumab in GCA. As a comparison, immune-inflammatory markers were also measured in sera from age- and sex-matched healthy volunteers. The biomarkers were quantified using luminex technology., Results: Of all the parameters determined, only MMP-3, pentraxin-3 and sTNFR2 were significantly elevated, while ICAM-1 and CD163 were significantly decreased during the early stages of the study, at time points of full clinical remission under treatment with tocilizumab plus glucocorticoids. In contrast, tocilizumab monotherapy towards the end of the study resulted in an almost complete normalization of immune-inflammatory molecules, as defined by the healthy controls. MMP-3 levels showed a weak association with magnetic resonance signal intensity; none of the biomarkers predicted relapse occurring within 6 months after study end., Conclusion: The data documented a subclinical disease activity in GCA that was more pronounced during the early stages of treatment and almost disappeared towards the study end. They indicated that tocilizumab treatment of at least 52 weeks is necessary in order to reset a broad range of immune-inflammatory pathways., Trial Registration: ClinicalTrials.gov, http://clinicaltrials.gov, NCT01450137.

Objective: To analyse magnetic resonance angiographic (MRA) vessel wall signals from a randomized controlled trial of tocilizumab (TCZ) to treat GCA., Methods: Participants were assigned in a 2:1 ratio to receive either TCZ + glucocorticoids (GCs) or placebo + GC infusions at 4-week intervals for 52 weeks. GCs were started at 1 mg/kg/day, then tapered to 0.1 mg/kg/day at week 12 and thereafter down to zero. Patients with initial positive MRA findings underwent control MRA at weeks 12 and 52. Vessel wall signals were scored from 0 (normal) to 3 (intense late enhancement). Outcomes were the number of patients with complete MRA remission at weeks 12 and 52, and changes in vasculitis score, vessel anatomy and atherosclerosis., Results: Of the 30 randomized participants, nine TCZ and two placebo patients had no vessel wall enhancement on initial MRA. At week 12, MRAs were performed in nine TCZ and four placebo patients (nine and three in clinical remission, respectively). Three (33%) TCZ patients showed normalization of vessel wall signals compared with one (25%) placebo patient. At week 52, there was additional MRA improvement in some TCZ patients, but one-third showed persistent or increased late vessel wall enhancement. There was no formation of aneurysms or stenosis and no increase in atherosclerosis., Conclusions: Although TCZ resulted in complete clinical and laboratory remission of GCA over 52 weeks, MRA signals in vessel walls normalized in only one-third of patients. Whether these signals are of prognostic importance remains to be determined.

Background: Giant cell arteritis is an immune-mediated disease of medium and large-sized arteries that affects mostly people older than 50 years of age. Treatment with glucocorticoids is the gold-standard and prevents severe vascular complications but is associated with substantial morbidity and mortality. Tocilizumab, a humanised monoclonal antibody against the interleukin-6 receptor, has been associated with rapid induction and maintenance of remission in patients with giant cell arteritis. We therefore aimed to study the efficacy and safety of tocilizumab in the first randomised clinical trial in patients with newly diagnosed or recurrent giant cell arteritis., Methods: In this single centre, phase 2, randomised, double-blind, placebo-controlled trial, we recruited patients aged 50 years and older from University Hospital Bern, Switzerland, who met the 1990 American College of Rheumatology criteria for giant cell arteritis. Patients with new-onset or relapsing disease were randomly assigned (2:1) to receive either tocilizumab (8 mg/kg) or placebo intravenously. 13 infusions were given in 4 week intervals until week 52. Both groups received oral prednisolone, starting at 1 mg/kg per day and tapered down to 0 mg according to a standard reduction scheme defined in the study protocol. Allocation to treatment groups was done using a central computerised randomisation procedure with a permuted block design and a block size of three, and concealed using central randomisation generated by the clinical trials unit. Patients, investigators, and study personnel were masked to treatment assignment. The primary outcome was the proportion of patients who achieved complete remission of disease at a prednisolone dose of 0·1 mg/kg per day at week 12. All analyses were intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01450137., Results: Between March 3, 2012, and Sept 9, 2014, 20 patients were randomly assigned to receive tocilizumab and prednisolone, and ten patients to receive placebo and glucocorticoid; 16 (80%) and seven (70%) patients, respectively, had new-onset giant cell arteritis. 17 (85%) of 20 patients given tocilizumab and four (40%) of ten patients given placebo reached complete remission by week 12 (risk difference 45%, 95% CI 11-79; p=0·0301). Relapse-free survival was achieved in 17 (85%) patients in the tocilizumab group and two (20%) in the placebo group by week 52 (risk difference 65%, 95% CI 36-94; p=0·0010). The mean survival-time difference to stop glucocorticoids was 12 weeks in favour of tocilizumab (95% CI 7-17; p<0·0001), leading to a cumulative prednisolone dose of 43 mg/kg in the tocilizumab group versus 110 mg/kg in the placebo group (p=0·0005) after 52 weeks. Seven (35%) patients in the tocilizumab group and five (50%) in the placebo group had serious adverse events., Interpretation: Our findings show, for the first time in a trial setting, the efficacy of tocilizumab in the induction and maintenance of remission in patients with giant cell arteritis., Funding: Roche and the University of Bern., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple osteochondromas (also called hereditary multiple exostoses) is an autosomal dominant disorder characterized by multiple cartilaginous tumors, which are caused by mutations in the genes for exostosin-1 (EXT1) and exostosin-2 (EXT2). The goal of this study was to elucidate the genetic alterations in a family with three affected members. Isolation of RNA from the patients' blood followed by reverse transcription and PCR amplification of selected fragments showed that the three patients lack a specific region of 90 bp from their EXT1 mRNA. This region corresponds to the sequence of exon 8 from the EXT1 gene. No splice site mutation was found around exon 8. However, long-range PCR amplification of the region from intron 7 to intron 8 indicated that the three patients contain a deletion of 4318 bp, which includes exon 8 and part of the flanking introns. There is evidence that the deletion was caused by non-homologous end joining because the breakpoints are not located within a repetitive element, but contain multiple copies of the deletion hotspot sequence TGRRKM. Exon 8 encodes part of the active site of the EXT1 enzyme, including the DXD signature of all UDP-sugar glycosyltransferases. It is conceivable that the mutant protein exerts a dominant negative effect on the activity of the EXT glycosyltransferase since it might interact with normal copies of the enzyme to form an inactive hetero-oligomeric complex. We suggest that sequencing of RNA might be superior to exome sequencing to detect short deletions of a single exon.

Mutations of STAT3 underlie the autosomal dominant form of hyperimmunoglobulin E syndrome (HIES). STAT3 has critical roles in immune cells and thus, hematopoietic stem cell transplantation (HSCT), might be a reasonable therapeutic strategy in this disease. However, STAT3 also has critical functions in nonhematopoietic cells and dissecting the protean roles of STAT3 is limited by the lethality associated with germline deletion of Stat3. Thus, predicting the efficacy of HSCT for HIES is difficult. To begin to dissect the importance of STAT3 in hematopoietic and nonhematopoietic cells as it relates to HIES, we generated a mouse model of this disease. We found that these transgenic mice recapitulate multiple aspects of HIES, including elevated serum IgE and failure to generate Th17 cells. We found that these mice were susceptible to bacterial infection that was partially corrected by HSCT using wild-type bone marrow, emphasizing the role played by the epithelium in the pathophysiology of HIES.

For acutely lethal influenza infections, the relative pathogenic contributions of direct viral damage to lung epithelium versus dysregulated immunity remain unresolved. Here, we take a top-down systems approach to this question. Multigene transcriptional signatures from infected lungs suggested that elevated activation of inflammatory signaling networks distinguished lethal from sublethal infections. Flow cytometry and gene expression analysis involving isolated cell subpopulations from infected lungs showed that neutrophil influx largely accounted for the predictive transcriptional signature. Automated imaging analysis, together with these gene expression and flow data, identified a chemokine-driven feedforward circuit involving proinflammatory neutrophils potently driven by poorly contained lethal viruses. Consistent with these data, attenuation, but not ablation, of the neutrophil-driven response increased survival without changing viral spread. These findings establish the primacy of damaging innate inflammation in at least some forms of influenza-induced lethality and provide a roadmap for the systematic dissection of infection-associated pathology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nuclear factor-κB (NF-κB) controls genes involved in normal lymphocyte functions, but constitutive NF-κB activation is often associated with B cell malignancy. Using high-throughput whole transcriptome sequencing, we investigated a unique family with hereditary polyclonal B cell lymphocytosis. We found a novel germline heterozygous missense mutation (E127G) in affected patients in the gene encoding CARD11, a scaffolding protein required for antigen receptor (AgR)-induced NF-κB activation in both B and T lymphocytes. We subsequently identified a second germline mutation (G116S) in an unrelated, phenotypically similar patient, confirming mutations in CARD11 drive disease. Like somatic, gain-of-function CARD11 mutations described in B cell lymphoma, these germline CARD11 mutants spontaneously aggregate and drive constitutive NF-κB activation. However, these CARD11 mutants rendered patient T cells less responsive to AgR-induced activation. By reexamining this rare genetic disorder first reported four decades ago, our findings provide new insight into why activating CARD11 mutations may induce B cell expansion and preferentially predispose to B cell malignancy without dramatically perturbing T cell homeostasis.

Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder characterized by cutaneous fibrofolliculomas, pulmonary cysts, and kidney malignancies. Affected individuals carry germ line mutations in folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney cancer, FLCN has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germ line deletion of Flcn results in early embryonic lethality in animal models. Here, we describe mice deficient in the newly characterized folliculin-interacting protein 1 (Fnip1). In contrast to Flcn, Fnip1(-/-) mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is entirely independent of mTOR activity. We show that this developmental arrest results from rapid caspase-induced pre-B cell death, and that a Bcl2 transgene reconstitutes mature B-cell populations, respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1(-/-) mice. Our studies thus demonstrate that the FLCN-FNIP complex deregulated in BHD syndrome is absolutely required for B-cell differentiation, and that it functions through both mTOR-dependent and independent pathways.

Distinct CD4(+) T cell subsets are critical for host defense and immunoregulation. Although these subsets can act as terminally differentiated lineages, they have been increasingly noted to demonstrated plasticity. MicroRNAs are factors that control T cell stability and plasticity. Here we report that naturally occurring regulatory T cells (T(reg) cells) had high expression of the microRNA miR-10a and that miR-10a was induced by retinoic acid and transforming growth factor-β (TGF-β) in inducible T(reg) cells. By simultaneously targeting the transcriptional repressor Bcl-6 and the corepressor Ncor2, miR-10a attenuated the phenotypic conversion of inducible T(reg) cells into follicular helper T cells. We also found that miR-10a limited differentiation into the T(H)17 subset of helper T cells and therefore represents a factor that can fine-tune the plasticity and fate of helper T cells.

The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy.

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity's microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Despite its clinical significance, the mechanism of progression into LBC is unknown. Here, we show that LBC but not CML cells express the B cell-specific mutator enzyme AID. We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.

We have identified a distinct pre-naive B cell population circulating in human peripheral blood that exhibits an intermediate phenotype between transitional and naive B cells. Like human transitional B cells, these cells express CD5 but have intermediate densities of CD38, CD10, CD9, and the ABCB1 transporter compared with transitional and naive B cells. These pre-naive B cells account for a majority of circulating human CD5(+) B cells. Importantly, CD5(+) pre-naive B cells could be induced to differentiate into cells with a naive phenotype in vitro. CD5(+) pre-naive B cells show only partial responses to BCR stimulation and CD40 ligation and undergo more spontaneous apoptosis and cell death than do naive B cells, whereas BAFF/BLyS (B cell-activating factor belonging to the TNF family) did not enhance their survival compared with naive B cells. In contrast, CD5(+) pre-naive B cells carry out certain functions comparable to naive B cells, including the capacity to differentiate into plasma cells and the ability to function as APCs. Notably, an increased proportion of CD5(+) pre-naive B cells were found in peripheral blood of patients with systemic lupus erythematosus. These results have identified a unique intermediate in human naive B cell development within the peripheral blood and derangements of its homeostasis in patients with systemic lupus erythematosus.

Summary: Interleukin-21 (IL-21) belongs to a family of cytokines that includes IL-2, IL-4, IL-7, IL-9, and IL-15, all of which bind to private (or shared) receptors as well as the common cytokine receptor gamma-chain as a component. Most cytokines in this family are critically important for both the maintenance and function of T cells and B cells. The receptor for IL-21 is widely distributed on lymphohematopoietic cells, and IL-21 plays many biologic roles, including maintenance and function of CD8(+) memory T cells and natural killer cells, as well as promoting the generation of Th17 cells in the mouse. One principal non-redundant role of IL-21 is the promotion of B-cell activation, differentiation or death during humoral immune responses. Furthermore, increased IL-21 production is characteristic of certain autoimmune diseases and is likely to contribute to autoantibody production as well as pathologic features of autoimmune disease. In contrast, IL-21 may function as a co-adjuvant to enhance antibody responses and thereby facilitate host defense to malignances and infectious diseases. The critical role of IL-21 in promoting humoral immune responses makes it an important focus of potential therapeutic interventions in conditions characterized by either overproduction of pathogenic autoantibodies or under production of protective antibodies.

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4(+) T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4(+) T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.

Both constitutive Ig secretion by long-lived plasma cells (PC) and the recurrent differentiation of memory (mem) B cells into PC contribute to the maintenance of serologic mem. However, the relative contribution of each is unknown. In this study, we describe a novel population of human postswitched mem B cells that rapidly differentiate into PC and thus contribute to serologic mem. These IgG(+) B cells reside in the region of human spleen analogous to the murine marginal zone and have not previously been examined. These cells are highly responsive to IL-21 in the context of CD40 stimulation. Uniquely, IgG(+) marginal zone analog B cells are exquisitely sensitive to the combination of IL-21 and B cell-activating factor belonging to the TNF family (BAFF/BLyS) that synergize in the absence of further costimulation to induce up-regulation of B lymphocyte-induced maturation protein-1 and drive PC differentiation. Other cytokine combinations are not active in this regard. This is the first demonstration that this unique population of mem B cells can respond specifically and exclusively to IL-21 and BAFF/BLyS by differentiating into IgG-secreting PC, and thus contributing to serologic mem in an Ag-independent manner.

The chapter focuses on the detection of specific mRNA by in situ hybridization (ISH) in synovial tissue specimens. This technique is widely applied, reliable, specific, and sensitive, because even small quantities of mRNA can be detected. Presented here contemporary protocols for ISH using a combined nonradioactive immunohistochemical detection system. In overview, the following steps have to be covered to perform ISH. (1) mRNA probes (sense and antisense) are generated by in vitro transcription of cDNA utilizing digoxigenin-labeled UTP nucleotides, (2) fixed tissue sections are digested with trypsin and treated consecutively with prehybridization solutions, (3) hybridization with labeled riboprobes takes place at 50 degrees C overnight in a humid chamber, (4) unbound riboprobe is removed by incubation with RNase A and additional washing with buffers, (5) stringent washing steps are performed with solutions of different sodium dodecyl sulfate, SSC, and formamide concentrations, (6) digoxigenin-labeled probes are detected immunohistochemically using antidigoxigenin antibodies linked with alkaline phosphatase and NBT/BCIP as detection system.

Objective: To study the specific contribution of MAP kinase activator c-Raf-1 and one of its downstream transcription factors, c-Myc, to the growth and invasive behavior of rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: RASFs were transduced with retroviral constructs expressing dominant-negative mutants of c-Raf-1 or c-Myc (DN c-Raf-1 or DN c-Myc, respectively) or with the mock vector. The expression of wild-type and mutant proteins was confirmed by Western blotting. Growth curves of RASFs were recorded, and apoptosis was measured by flow cytometry. Invasiveness of RASFs was assessed in the SCID mouse model of RA. Immunohistochemistry was used to study the effects of DN c-Raf-1 on phosphorylated c-Jun and matrix metalloproteinase 1 (MMP-1) in RASFs implanted into SCID mice. The phosphorylation of ERK and JNK in DN c-Raf-1- and mock-transduced RASFs was determined in vitro by Western blotting. The levels of MMPs in these cells were measured by quantitative polymerase chain reaction (PCR)., Results: Neither DN c-Raf-1 alone nor DN c-Myc alone significantly altered proliferation or apoptosis of RASFs, but both mutants together rapidly induced apoptosis. Inhibition of c-Raf-1 or c-Myc significantly reduced the invasiveness of RASFs in the SCID mouse model. DN c-Raf-1 decreased the phosphorylation of ERK and JNK in vitro and reduced the in vivo expression of phosphorylated c-Jun as well as the expression of disease-relevant MMPs. As determined by quantitative PCR, the inhibition was most pronounced for MMP-1 and MMP-3., Conclusion: The data demonstrate that Ras- and c-Myc-dependent signaling events cooperate to regulate the growth and invasiveness of RASFs. Targeting of both c-Raf-1 and c-Myc may constitute an interesting therapeutic approach in RA.

Objective: To describe cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) expression in muscle tissue in patients with idiopathic inflammatory myopathies (IIM) - dermatomyositis (DM) and polymyositis (PM) and to find out if any differences between affected and non-affected muscles detected by MRI exist., Methods: Samples of muscle tissue from 7 patients with dermatomyositis (DM) and from 4 with polymyositis (PM) were obtained by needle biopsy from affected and non-affected sites distinguished by magnetic resonance imaging. In situ hybridization with antisense mRNA probes was employed to detect COX-1, COX-2 and 5-LOX mRNA., Results: Expression of COX-1, COX-2, and 5-LOX mRNA was found in all samples - in the muscle cells, inflammatory cells and in vessels. COX-1 mRNA expression predominated in the inflammatory cells and vessels and was higher in affected than in non-affected sites detected by MRI (mean intensity 3.22+/-0.67 vs. 2.0+/-0.87; p = 0.0006). The expression of COX-2 mRNA was high mainly in inflammatory cells and/or vessels and was increased in MRI-detected affected tissues (3.5+/-0.88; 1.9+/-1.1; p = 0.003), as was the expression of COX-2 mRNA in muscle cells (2.1+/-1.0 vs. 1.3+/-1.0; p = 0.021). 5-LOX mRNA was largely expressed in muscle cells from MRI-detected affected sites and the signal intensity was higher in comparison with samples taken from non-affected tissues detected by MRI (3.22+/-0.7 vs. 1.67+/-0.7; p = 0.0007)., Conclusion: Expression of COX-1, COX-2 and 5-LOX mRNA was observed for the first time in muscle tissues from IIM patients. This expression was increased in affected tissues detected by MRI, which may suggest a role of COX-1, COX-2, and 5-LOX in the pathogenesis of IIM.

We characterized a full length L1 mRNA in a rheumatoid arthritis (RA) synovial tissue and determined the degree of methylation of its 5'-UTR. We asked whether not only intact but also altered L1s can exert biological activities by transfecting RA synovial fibroblasts (SF) with either retrotransposition-competent or incompetent L1s and examined their capacity to induce p38delta. Total RNA was isolated from the synovial tissue of a 35-year-old woman with highly destructive RA. A complete L1 sequence was obtained by 3'/5'-RACE. Methylation of the genomic 5'-UTR was determined by the sodium-disulfide/PCR method. RA-SF were transfected by lipofection with either a functional L1 or an ORF2-mutated L1 element. The expression of p38delta was measured by RT-PCR and Western blot. The full length L1 mRNA included a 5'-UTR, an ORF1 and an ORF2. Three of five CpG islands (60%) of the genomic L1 5'-UTR were hypomethylated and the ORF2 was deactivated by the insertion of stop codons. Both, intact and ORF2-mutated L1 vectors, induced the expression of p38delta. Thus, even an ORF2-mutated L1 element, as expressed in RA, is biologically active and both L1 ORF1 and p38delta transcripts may appear as a consequence of genomic hypomethylation. The induction of p38delta appears to be mediated by an ORF1/p40-dependent process. This is the first indication of a p40 mediated transactivation.

Objective: To characterize the expression pattern and role of galectin 3 and galectin 3 binding protein (G3BP) in rheumatoid arthritis (RA), in comparison with galectin 1, and to explore whether soluble galectin 3 and G3BP, investigated in serum, synovial fluid, or cell culture supernatant, are associated with disease., Methods: Synovial tissues from patients with RA or osteoarthritis (OA), as well as from healthy controls, were analyzed for galectins 1 and 3 and G3BP by in situ hybridization and immunohistochemistry. Levels of galectin 3 and G3BP in serum and synovial fluid from patients with RA and OA and controls, as well as in cell culture supernatants, were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the intracellular expression of galectin 3 in RA and OA synovial fibroblasts after modulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and anti-CD40 monoclonal antibodies was measured by flow cytometry., Results: In RA, galectin 3 messenger RNA and protein stained throughout the synovial membrane, whereas G3BP was particularly expressed at sites of bone destruction. In contrast, the expression of galectin 1 was not uniform in different RA specimens, and was never found at sites of invasion. In OA and normal synovial tissues, only a small number of cells were positive for galectins and/or G3BP. Galectin 3 was elevated in RA sera and synovial fluids, whereas G3BP was increased in RA synovial fluids only. In RA, serum galectin 3 correlated with C-reactive protein levels, whereas G3BP was associated with joint destruction and/or synovial cell activation as measured by the levels of cartilage oligomeric matrix protein. In vitro, RA synovial fibroblasts showed an increased release of galectin 3 into culture medium, as measured by ELISA, but decreased secretion of G3BP. In RA synovial fibroblasts with low basal expression of galectin 3, TNFalpha increased its intracellular level in a dose-dependent manner. In contrast, IL-1beta or anti-CD40 monoclonal antibodies showed no effect., Conclusion: Our data indicate that galectin 3 and G3BP are not only involved in inflammation, but also contribute to the activation of synovial fibroblasts. The intracellular accumulation of galectin 3 can be enhanced by TNFalpha. Thus, galectin 3 and G3BP represent novel markers of disease activity in RA.

The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.