ABSTRACT
Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.
Subject(s)
C-Reactive Protein/genetics , Macrophages, Peritoneal/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Opsonin Proteins/genetics , Phagocytosis/genetics , Serum Amyloid P-Component/genetics , Zymosan/immunology , Animals , Binding Sites/drug effects , Binding Sites/genetics , C-Reactive Protein/metabolism , Female , Immunity, Innate/genetics , Lectins, C-Type , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Male , Membrane Glycoproteins/drug effects , Membrane Glycoproteins/metabolism , Membrane Proteins/antagonists & inhibitors , Mice , Mice, Transgenic , Nerve Tissue Proteins/antagonists & inhibitors , Nitric Oxide/metabolism , Opsonin Proteins/metabolism , Paracoccidioides/immunology , Phagocytosis/drug effects , Phagocytosis/immunology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/metabolism , Serum Amyloid P-Component/metabolism , Toll-Like Receptor 6 , Zymosan/metabolism , Zymosan/pharmacologyABSTRACT
Using DDRT-PCR, we compared the mRNA content of untreated and TNF-treated mouse embryonic fibroblasts (MEFs). Among differentially represented fragments, we identified and cloned a novel TNF-stimulated gene named Tsg-5. This gene, mapped to mouse chromosome 14, has three exons that can be alternatively spliced giving rise to two mRNA species, one spanning three exons and another that skips the second exon. Analysis of full-length Tsg-5 cDNA revealed a potential start codon within exon 2 encoding an ORF of 40 amino-acids. No homology with known mouse or human sequences, neither at the nucleotide nor at the amino-acid level could be found in public databases. In MEFs, Tsg-5 is induced by tumor necrosis factor-alpha (TNF) and IL-1 beta, albeit with distinct kinetics. TNF-induced Tsg-5 expression is NF-kappa B-dependent as it was inhibited by MG132, lactacystin, Bay 11-7083, and Bay 11-7085. Analysis of Tsg-5 expression in vivo revealed that the gene and its encoded polypeptide are constitutively expressed in the thymus and ovary, whereas, in LPS-treated mice, Tsg-5 mRNA can be detected in the spleen, lung, and brain. Our data suggest that Tsg-5 encodes a new, rare transcript, with a very tight regulation of expression and differential splicing.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Tumor Necrosis Factor-alpha/physiology , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/isolation & purification , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/physiology , Female , Gene Expression Regulation/immunology , Interferon Regulatory Factor-1 , Mice , Mice, Inbred BALB C , Mice, Knockout , Molecular Sequence Data , NF-kappa B/metabolism , Organ Specificity/genetics , Organ Specificity/immunology , Phosphoproteins/biosynthesis , Phosphoproteins/deficiency , Phosphoproteins/genetics , Polymerase Chain ReactionABSTRACT
OBJECTIVE: Expression of TSG-6 (tumor necrosis factor-stimulated gene 6) is induced by proinflammatory cytokines. This study was undertaken to examine the effects of local expression of TSG-6 in arthritic joints of TSG-6 transgenic mice, in the collagen-induced arthritis (CIA) model. METHODS: We generated transgenic mice that harbored the TSG-6 gene under the control of the T cell-specific lck promoter. Arthritis was induced by immunization with bovine type II collagen (CII), and its progression was monitored based on the incidence of arthritis, the arthritis index, and footpad swelling. Anti-CII antibodies and cytokine production were determined by enzyme-linked immunosorbent assay. Gene expression arrays were used to compare gene expression profiles of transgenic and control mice at various stages of CIA. RESULTS: TSG-6 was expressed in limbs of transgenic mice after immunization with CII, while its expression in nontransgenic animals was insignificant. The incidence of CIA was reduced in TSG-6 transgenic animals, its onset delayed, and all parameters of clinical arthritis significantly reduced. However, the immune response against CII was not significantly inhibited in TSG-6 transgenic mice. CONCLUSION: TSG-6 expression has been demonstrated in patients with rheumatoid and other forms of arthritis. Our data show that local expression of TSG-6 at sites of inflammation results in potent inhibition of inflammation and joint destruction in a model of autoimmune arthritis in mice. Therefore, it is likely that TSG-6 plays a similar modulatory role in human rheumatoid arthritis and related diseases and may have potential for the treatment of autoimmune arthritis in humans.
Subject(s)
Arthritis/chemically induced , Arthritis/genetics , Cell Adhesion Molecules/genetics , Collagen Type II , Genetic Predisposition to Disease/genetics , Animals , Antibody Formation , Arthritis/pathology , Arthritis/physiopathology , Cattle , Cell Division/drug effects , Cells, Cultured , Collagen Type II/immunology , Collagen Type II/pharmacology , Cytokines/biosynthesis , Gene Expression , Joints/physiopathology , Mice , Mice, Inbred DBA , Mice, Transgenic/genetics , T-Lymphocytes/pathology , TransgenesABSTRACT
The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31 percent) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38 percent) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs
