Abstract: Tumor necrosis factor (TNF)-alpha inhibitors are increasingly being used to treat rheumatoid arthritis. Infliximab (INF) is a TNF-alpha inhibitor that is usually used in combination with methotrexate (MTX). Interstitial lung disease (ILD) during combination therapy has been attributed to MTX rather than INF. However, INF-associated ILD without combination with MTX has recently been reported. We describe herein a case of severe ILD secondary to INF without MTX therapy.
Abstract: Hemophagocytic syndrome (HPS) is an unusual but sometimes fatal disorder. We reported a case of 21-year-old man who developed HPS and SLE simultaneously. Febrile pancytopenia, hyperferritinemia, and abnormal liver function tests were observed. Hemophagocytic cells were observed by means of bone marrow biopsy and diagnosed as HPS. The patient was treated with high-dose prednisolone, resulting in an excellent outcome. Early diagnosis of HPS by bone marrow biopsy is important for the successful treatment.
Abstract: The aim of the present study was to assess the influence of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis. Changes in clinical variables and radiological findings in 213 weight-bearing joints (69 hip joints, 63 knee joints, and 81 ankle joints) of 42 consecutive patients were investigated at baseline and at 1 year of TNF-blocking therapies. Structural damage to the weight-bearing joints was assessed using the Larsen scoring method. Detailed comparisons of the sizes and locations of erosions were performed for each set of radiographs of the respective joints. Assessment of radiographs of the 213 weight-bearing joints indicated progression of the Larsen grade in eight joints. Another five joints without Larsen grade progression showed apparent radiographic progression of joint damage based on increases in bony erosions. Overall, 13 joints (6%) of eight patients (19%) showed progression of joint damage after 1 year of TNF-blocking therapies. Analysis of each baseline grade indicated that radiographic progression of joint damage was inhibited in most grade 0-II joints. On the other hand, all hip and knee joints with pre-existing damage of grade III/IV showed apparent progression even in patients with good response. The results further suggested that radiographic progression may occur in less damaged joints when the patients were non-responders to the therapy. Among the weight-bearing joints, ankle joints showed different radiographic behavior and four ankle joints displayed improvement of radiographic damage. Early initiation of anti-TNF therapy should be necessary especially when the patients are starting to show early structural damage in weight-bearing joints.
Abstract: A 23-year-old Japanese man was admitted to our hospital because of dry cough and lacrimal gland swelling. Laboratory findings showed hypergammaglobulinemia (4859 mg/dl), hypocomplementemia (CH50 13 U/ml), and hyperamylasemia. CT revealed a marked swelling of the bilateral lacrimal glands, diffuse patchy infiltration in the bilateral lung fields, and enlargement of the whole pancreas. Gallium citrate scintigraphy showed abnormal accumulation in the bilateral lacrimal glands, submandibular glands, and both lung fields. Biopsy specimens from the salivary gland revealed dense lymphoplasmacytic infiltration and abundant IgG4-positive plasma cells. Furthermore, open lung biopsy showed a marked peribronchial lymphoplasmacytic and eosinophilic infiltration with IgG4-positive plasma cells. These findings fulfilled the criteria of Mikulicz's disease and autoimmune pancreatitis, and support the recently proposed concept of IgG4-related systemic disease.
Abstract: Tumor necrosis factor-alpha (TNF-alpha) plays critical roles in bone resorption at the site of inflammatory joints. The aim of this study is to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists, a new class of anti-inflammatory compounds, on TNF-alpha-mediated osteoclastogenesis in human monocytes. Human monocytes were differentiated into osteoclasts in the presence of TNF-alpha and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay using dentin were used for the identification of activated osteoclasts. The protein and gene expressions of transcription factors were determined by immunofluorescence and real-time RT-PCR analysis, respectively. TNF-alpha-induced osteoclast generation from human peripheral monocytes in a dose-dependent manner, and the induction was not inhibited by osteoprotegerin, a decoy receptor for receptor activator of NF-kappaB ligand. The addition of PPAR-gamma agonists, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, to the culture resulted in a remarkably reduced number of generated osteoclasts. In addition, both agonists inhibited the protein and gene expressions of nuclear factor of activated T-cell isoform c1 (NFATc1), c-Fos, c-Jun and NF-kappaB p65, which are known to be associated with osteoclastogenesis. GW9662, an antagonist of PPAR-gamma, fully rescued ciglitazone-induced inhibition, but did not affect 15d-PGJ2-induced inhibition. Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine related to osteoclastogenesis, was induced during TNF-alpha-mediated osteoclast differentiation, and the neutralizing antibody to MCP-1 reduced osteoclast formation by about 40%. 15d-PGJ2 and ciglitazone blocked the induction of MCP-1 by TNF-alpha. Moreover, the addition of MCP-1 rescued the inhibition of TRAP-positive multinucleated cell (TRAP-MNCs) formation by 15d-PGJ2 and ciglitazone, although generated TRAP-MNCs had no capacity to resorb dentin slices. Our data demonstrate that 15d-PGJ2 and ciglitazone down-regulate TNF-alpha-mediated osteoclast differentiation in human cells, in part via suppression of the action of MCP-1. These PPAR-gamma agonists may be a promising therapeutic application for rheumatoid arthritis and inflammatory bone-resorbing diseases.
Abstract: Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.
Abstract: We present a case of thymic carcinoid, in which primary and metastatic lesions of lymph nodes and bones could be detected by [(18)F]fluoro-2-deoxy-D-glucose (FDG)-PET, but not by (123)I-meta-iodobenzylguanidine ((123)I-MIBG) SPECT, or by (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) bone scintigraphy. FDG-PET may be a useful tool for managing thymic carcinoids in patients with negative results on (123)I-MIBG SPECT or (99m)Tc-MDP imaging.
Abstract: Interleukin-10 (IL-10), an anti-inflammatory cytokine, has been shown to inhibit osteoclast formation and bone resorption in rat and mouse systems. However, the precise intracellular mechanism(s) of this action remains unclear. The aim of this study was to clarify the role of IL-10 in the regulation of critical transcription factors involved in osteoclastogenesis. A RAW264.7 macrophage cell line, which constitutively expressed IL-10 receptor, was differentiated to osteoclasts with stimulation of receptor activator of nuclear factor kappaB ligand (RANKL). IL-10 inhibited the RANKL-induced osteoclastogenesis. IL-10 potently reduced the RANKL-induced expression of NFATc1, c-Jun and c-Fos, which are known to be essential for osteoclastogenesis, in time- and dose-dependent manners. The IL-10-induced inhibition of these transcription factors was observed in the system of mouse bone marrow precursors. Besides these transcription factors, IL-10 also decreased the RANKL-induced expression of NF-kappaB p50 and phosphorylation of JNK. To determine which signaling was critical for the IL-10 effect, we examined the effect of overexpression of NFATc1, c-Fos, and c-Jun on the IL-10-induced inhibition of osteoclastogenesis. As expected, overexpression of NFATc1 abrogated the IL-10-induced inhibition of osteoclastogenesis. Interestingly, overexpression of either c-Fos or c-Jun partially rescued the reduction of RANKL-induced expression of NFATc1 and osteoclastogenesis by IL-10. These data suggest that IL-10 may down-regulate osteoclastogenesis mainly through inhibition of the expression of NFATc1, c-Fos and c-Jun. These findings provide new insight into the inhibitory action of IL-10 on RANKL-mediated osteoclastogenesis.
Abstract: Interleukin-4 (IL-4), an anti-inflammatory cytokine, has been shown to inhibit osteoclast differentiation. Therefore, this cytokine is considered to be a promising therapeutic applicant for bone-resorbing diseases such as rheumatoid arthritis (RA). Recently NFATc1, a transcription factor, has been shown to play critical roles in osteoclastogenesis. The aim of this study was to clarify the role of IL-4 on the intracellular signaling of NFATc1. A RAW264.7 monocyte/macrophage cell line and murine bone marrow precursors were differentiated into osteoclasts in the presence of receptor activator of nuclear factor kappaB ligand (RANKL) and/or macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine were used for the identification of activated osteoclasts. The protein expression of IL-4 receptor, NFATc1, and c-Fos was determined by Western blot analysis. In addition, the gene expression of NFATc1 and c-Fos was determined by reverse transcription and polymerase chain reaction. The IL-4 receptor was constitutively expressed in RAW264.7 cells. RANKL induced osteoclast generation, as determined by TRAP staining and pit assay. IL-4 inhibited RANKL-induced osteoclastogenesis at low concentrations of 10ng/ml and more. Interestingly, IL-4 potently inhibited RANKL-induced expression of NFATc1 at mRNA level. Furthermore, IL-4 inhibited c-Fos expression, which is shown to be responsible for NFATc1 expression, in time- and dose-dependent manners. In addition, IL-4 inhibited the RANKL-induced expression of NFATc1 and c-Fos in murine bone marrow cells. Thus, we suggest that IL-4 may downregulate osteoclastogenesis in part through inhibition of the expression of transcription factors, NFATc1 and c-Fos. These findings provide new insight into development of new medication for osteoporosis and RA.
Abstract: There is accumulating evidence that T cells may be involved in osteoclastogenesis in a variety of murine systems. However, the precise role of human T cells in the regulation of osteoclast generation is still unclear. To address this issue, we investigated the effect of resting peripheral T cells on receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast generation from human peripheral monocytes. Although osteoclasts were not generated in the culture of human peripheral blood mononuclear cells (PBMC) in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), the addition of cyclosporine A (CsA), a potent inhibitor of T-cell function, resulted in the formation of an increasing number of lacunae resorption on dentine, suggesting T cells may inhibit osteoclast formation. In a coculture of T cells and monocytes, which were isolated from PBMC, T cells inhibited the osteoclast generation from monocytes, as determined by tartrate-resistant acid phosphatase (TRAP) staining and a pit assay using dentine. This inhibition of osteoclast generation by T cells was also observed in a culture of the parathyroid hormone-stimulated SaOS4/3 osteoblast cell line and monocytes. The culture in Transwell plates revealed that the cell-to-cell interaction was not required for the inhibition, suggesting that T-cell cytokines may be responsible for the inhibition. Among inhibitory T-cell cytokines on osteoclastogenesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) were actively produced by CD4 T cells but not CD8 T cells in the coculture of T cells with monocytes, and the neutralizing antibodies to these cytokines partially rescued the T-cell-induced inhibition of osteoclast formation. Although CsA did not affect RANKL-induced osteoclast generation in the culture of monocytes alone, it completely rescued the T-cell-induced inhibition of osteoclast formation and strongly inhibited the production of GM-CSF and IFN-gamma. Thus, we demonstrate that resting T cells negatively regulate the osteoclast generation via production of GM-CSF and IFN-gamma by CD4 T cells and that CsA stimulates the osteoclast generation through the inhibition of the production of these cytokines. These findings provide new insight into therapeutic strategies for immunosuppression-induced bone loss in transplant and other diseases.
Abstract: OBJECTIVE: To clarify the role of interleukin-4 (IL-4) in the expression of 15-lipoxygenase (15-LOX), whose metabolities are known to suppress the inflammatory reaction, in freshly prepared rheumatoid synovial cells. METHODS: Adherent synovial cells were prepared by enzymatic digestion of synovia obtained from patients with rheumatoid arthritis (RA). Protein expression of 15-LOX was determined by Western blot analysis. The messenger RNAs of 15-LOX were determined by reverse transcription and the polymerase chain reaction (RT-PCR). RESULTS: Freshly prepared rheumatoid synovial cells did not express 15-LOX at either the mRNA or protein levels. IL-4 induced the protein expression of 15-LOX after 24 hours of culture. Although interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha), major inflammatory cytokines in rheumatoid synovia, did not induce the expression of 15-LOX, IL-4 and these inflammatory cytokines synergistically enhanced the protein expression of 15-LOX. The synergistic effect was also observed at the level of mRNA. CONCLUSIONS: We demonstrate that IL-4 cooperated with the inflammatory cytokines IL-1 alpha and TNF alpha to enhance the expression of 15-LOX in rheumatoid synovial cells. Since 15-LOX metabolites have potent anti-inflammatory actions, our data suggest that IL-4 might downregulate rheumatoid inflammation via the induction of 15-LOX and its metabolites.
Abstract: OBJECTIVE: D-penicillamine (DP) has been shown to cooperate with copper ion to inhibit cell growth in a variety of cell types. To determine whether this inhibitory action is involved in Fas-mediated apoptosis, we examined the effect of DP and copper sulfate on the expression and function of Fas antigen in rheumatoid synovial fibroblasts (RSFs). METHODS: The expression of Fas antigen on the cell surface was determined by flow cytometric analysis. Western blot analysis was performed to examine the protein expressions of Fas and Fas-ligand In addition, the amounts of apoptotic cells were determined by 4', 6-diamidino-2'-phenylindol dihydrochloride (DAPI) and propidium iodide (PI) staining. RESULTS: Although DP and copper sulfate alone did not affect the surface expression of Fas antigen on RSFs, both in combination augmented the Fas expression in dose- and time-dependent manners. The enhanced expression of Fas antigen on their surface was also observed in interleukin-1alpha (IL-1alpha) and/or tumor necrosis factor a (TNFalpha) stimulated RSFs. On the other hand, the combination of DP and copper sulfate did not increase the amounts of cellular Fas protein, as determined by Western blot analysis. To determine whether the induced Fas antigen is functional, we examined the effect of DP and copper sulfate on Fas-mediated apoptosis, using an agonistic anti-Fas antibody. The treatment of this antibody induced the apoptosis in untreated RSFs, as determined by DAPI staining. The combination of DP and copper sulfate further enhanced the Fas-mediated apoptosis. The enhanced apoptosis and cell surface expression of Fas was completely prevented by catalase, indicating that hydrogen peroxide may be involved in these effects of DP and copper sulfate. The protein expression of Fas-ligand, a natural ligand for Fas antigen, in RSFs. was expressed in untreated RSFs. However, the protein levels were not modulated by DP and copper sulfate. CONCLUSIONS: Our data demonstrated that DP cooperated with copper sulfate to enhance the cells surface expression of functional Fas antigen in RSFs. In addition, Fas-ligand was expressed in the RSFs. These findings suggested that DP might regress rheumatoid synovial hyperplasia via Fas-mediated apoptosis.
Abstract: We describe a patient with hereditary angioedema (HAE), showing recurrent edema around the peripheral joints. Her symptoms began at the age of 18 with hand swelling distal to the wrist joints. Until she was referred to our hospital 3 years after her initial symptoms, she was still undiagnosed, although she was suspected of having rheumatoid arthritis. Laboratory examination showed reduced levels of CH50 and C4 with normal C3 levels. The C1 inhibitor (C1-INH) was decreased to 5 mg/ml, with remarkably reduced activity. Although these findings were compatible with a diagnosis of HAE, there were no episodes of skin edema in her family. To establish the diagnosis, we carried out DNA analysis of the C1-INH gene, which revealed a newly identified de novo mutation of G to A at nucleotide 16869 in exon 8. As described in this patient, localized edema around the peripheral joints may be the only manifestation of HAE. HAE should therefore be taken into consideration for the differential diagnosis of joint swelling.
Abstract: OBJECTIVE: To examine the effect of interleukin-4 (IL-4) on IL-11 production by rheumatoid synovial cells. METHODS: Freshly isolated rheumatoid synovial cells (FRS) were obtained by collagenase digestion of rheumatoid arthritis (RA) synovial tissue specimens taken at the time of operation. Rheumatoid synovial cells at four to eight passages were used as cultured rheumatoid synovial fibroblasts (RSF). IL-11 concentration was measured by ELISA. RESULTS: IL-4 inhibited the production of IL-11 by FRS in a dose-dependent manner. This inhibition was observed in FRS obtained from six patients, and the mean inhibition was 46.5%. The inhibitory effect of IL-4 on IL-11 production was cancelled by the addition of anti-IL-4 antibody. IL-4 also inhibited IL-11 production by IL-1alpha-stimulated cultured RSF. CONCLUSION: IL-4 inhibited IL-11 production by rheumatoid synovial cells. IL-4 has a protective effect on bone resorption. On the contrary, IL-11 participates in bone resorption via osteoclastogenesis. Therefore, IL-4 may exert its protective effect on bone resorption, at least in part, via inhibition of IL-11 production in rheumatoid joints.
Abstract: Human vascular smooth muscle cells (VSMC) are a component of blood vessels, and secrete a variety of cytokines in atherosclerotic loci. Interleukin-11 (IL-11), a member of IL-6-like cytokines, is reported to be involved in inflammation and tissue remodeling, both of which are observed in atherosclerosis. However, no information is available as to the production of IL-11 by VSMC. Therefore, the expression of IL-11 in VSMC is investigated. The amounts of IL-11 protein and mRNA were determined by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis, respectively. The expression of IL-11 in VSMC was also immunohistochemically determined. IL-1 alpha, transforming growth factor-beta (TGF beta) and, to a lesser extent, tumor necrosis factor-alpha (TNF alpha) stimulated the IL-11 production by VSMC, and the stimulatory effects of IL-1 alpha and TGF beta on IL-11 production were dose-dependent. IL-1 alpha and TNF alpha synergistically augmented TGF beta-stimulated IL-11 production by VSMC. Immunohistochemical staining also revealed the expression of IL-11 protein in VSMC. Furthermore, IL-1 alpha, TGF beta, and TNF alpha induced IL-11 gene expression in VSMC. Because IL-6-like cytokines are reported to be cytoprotective, monokine-stimulated IL-11 may have a potent protective role in atherosclerotic lesions.
Abstract: Interleukin-11 (IL-11) is known to be a member of the interleukin-6 (IL-6)-type cytokine family. IL-11 is likely to be a major determinant of immune regulation in acute and chronic inflammatory lung diseases, although it is not directly linked with specific disease processes. It has already been shown that although unstimulated human lung fibroblasts did not produce significant amounts of IL-11, the addition of interleukin-1 alpha (IL-1 alpha) and/or transforming growth factor-beta (TGF-beta) stimulated fibroblasts dose-dependently to produce IL-11. Northern blot analysis showed that these stimulators also upregulated IL-11 mRNA expression. As it has been previously reported that IL-1 and TGF-beta stimulate prostaglandin E2 (PGE2) release from lung fibroblasts, we investigate here the role of endogenous PGE2 and the direct effects of the two inhibitors of prostaglandin synthesis, indomethacin and dexamethasone, on IL-11 production by human lung fibroblasts. The addition of indomethacin, a cyclo-oxygenase inhibitor, resulted in significant suppression of IL-11 production and mRNA expression in lung fibroblasts. There was no detectable effect of PGE2 alone on IL-11 levels; however, the suppression of IL-11 production by indomethacin was almost completely reversed by addition of PGE2. In contrast, suppression of IL-11 production by indomenthacin was not reversed by addition of thromboxane B2 and carbocyclic thromboxane A2. In addition, dexamethasone completely suppressed IL-11 production and downregulated IL-11 mRNA. These results suggest that endogenous PGE2 acts as an autocrine stimulus for IL-11 production by human lung fibroblasts activated by IL-1 alpha and TGF-beta.
Abstract: OBJECTIVE: Interleukin-11 (IL-11), an IL-6-type cytokine, is thought to be involved in bone resorption via osteoclast differentiation. Here, we characterized the combined effect of IL-1alpha and tumor necrosis factor alpha (TNFalpha), major cytokines in the rheumatoid synovium, on the production of IL-11 by cultured rheumatoid synovial fibroblasts (RSFs). METHODS: The amounts of IL-11, IL-6, and prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay. IL-11 messenger RNA (mRNA) levels were determined by Northern blotting. Protein expression of cytosolic phospholipase A2 (cPLA2), cyclooxygenase 2 (COX-2), and protein kinase C (PKC) isoforms were determined by Western blotting. RESULTS: IL-1alpha and TNFalpha synergistically stimulated RSFs to produce IL-11 at both the mRNA and protein levels. This synergistic effect was completely inhibited by indomethacin. The inhibition was prevented by PGE2, indicating that the synergistic effect of IL-1alpha and TNFalpha was PGE2-mediated. The cooperative effects of these 2 cytokines were also observed in the production of PGE2 and the expression of 2 regulatory enzymes in PGE2 production, cPLA2 and COX-2. The synergistic induction of IL-11 by IL-1alpha and TNFalpha was completely inhibited by a potent inhibitor of all isoforms of PKC, GF109203X. In contrast, phorbol myristate acetate, which induced a down-regulation of PKC, degrading all PKC isoforms except atypical PKC, did not affect the induction of IL-11. CONCLUSION: These findings suggest that IL-1alpha and TNFalpha synergistically stimulate the production of IL-11 via their effects on PGE2 production in the rheumatoid joint, and that atypical PKC may be another target for down-regulation of IL-11, the bone resorption-associated cytokine.
Abstract: IL-11, a member of the IL-6 type cytokines, has some biological activity related to the joint destruction in rheumatoid arthritis (RA), such as induction of osteoclast differentiation. However, its expression and regulation in rheumatoid inflamed joints has not been clarified. In the present study we examined the capacity of fresh rheumatoid synovial cells (fresh RSC) to produce IL-11, and the effect of indomethacin, dexamethasone and IFN-gamma on IL-11 production. Fresh RSC obtained from eight patients with RA produced large amounts of IL-11, measured by ELISA, and showed strong expression of IL-11 mRNA, determined by Northern blotting. Indomethacin inhibited the production of IL-11 by about 55%. Prostaglandin E2 (PGE2) completely prevented the inhibition, suggesting that IL-11 production by fresh RSC was in part mediated by PGE2. Dexamethasone inhibited the production of IL-11 by more than 80%. Interestingly, the inhibition was not abolished by PGE2. IFN-gamma inhibited the production of IL-11 from IL-1alpha-stimulated cultured rheumatoid synovial fibroblasts, although IFN-gamma did not inhibit the production of IL-11 by fresh RSC. These results suggest that the production of IL-11 by rheumatoid synovia was differentially regulated by PGE2 and IFN-gamma, and that treatment with indomethacin or dexamethasone decreased the level of IL-11 at inflammatory joints in patients with RA.
Abstract: We recently reported that interleukin-4 (IL-4) inhibited prostanoid synthesis through inhibiting cyclooxygenase 2 biosynthesis. In the present study, we examined the effect of IL-4 on the expression of cytosolic phospholipase A2 (cPLA2). The amounts of protein and mRNA of cPLA2 were determined by western blotting and reverse transcription polymerase chain reaction (RT-PCR), respectively. Although interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha (TNFalpha) had little effect on the biosynthesis of cPLA2 in phorbol myristate acetate (PMA)-differentiated U937 cells, lipopolysaccharide (LPS) increased the protein level of cPLA2 in a dose-dependent manner. IL-4 inhibited the increased synthesis of cPLA2 at the mRNA level. In addition, IL-4 inhibited the biosynthesis of cPLA2 in untreated or LPS treated freshly prepared rheumatoid synovial cells at the mRNA level. These findings suggest that IL-4 inhibits prostanoid synthesis through inhibiting the expression of both cPLA2 and cyclooxygenase 2.
Abstract: n-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cells ex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1 beta (IL-1 beta) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fat-free balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk. spleen cell preparations were stimulated ex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and proIL-1 beta mRNA (IL-1 beta mRNA) was measured by northern analysis. Levels of IL-1 beta mRNA in both LPS- and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1 beta gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1 beta gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.
Abstract: OBJECTIVE: To characterise the effect of interleukin-4 (IL-4) on the biosynthesis of cyclo-oxygenases I (COX I) and II (COX II), the rate limiting enzymes of the synthesis of prostaglandin E2 (PGE2), in freshly prepared rheumatoid synovial cells. METHODS: Adherent synovial cells were obtained from rheumatoid synovium by collagenase digestion. The concentrations of PGE2 in culture supernatants were determined by enzyme linked immunosorbent assay. The protein and mRNA concentrations of COX I and COX II were determined by Western blotting and reverse transcription polymerase chain reaction, respectively. RESULTS: Freshly prepared synovial cells produced large amounts of PGE2. They also showed increased gene expression of COX I and COX II, and synthesised these proteins. IL-4 had suppressive effects on the production of PGE2 by untreated or lipopolysaccharide (LPS) stimulated synovial cells. In addition, IL-4 inhibited the biosynthesis of COX II at the mRNA level. In contrast, it did not modify the protein concentration of COX I. In tests of cell specificity, IL-4 did not reduce the mRNA concentration of COX II in interleukin-1 alpha (IL-1 alpha) stimulated cultured synovial fibroblasts at passages 3-6, but it reduced considerably the mRNA concentrations of COX II in an LPS or IL-1 alpha stimulated U937 monocyte/macrophage cell line. CONCLUSIONS: These results suggest that IL-4 might inhibit overproduction of PGE2 in rheumatoid synovia via selective inhibition of the biosynthesis of COX II, and that this inhibition might be specific to macrophage-like synovial cells.
Abstract: In rheumatoid synovium, interleukin-6 (IL-6) is the most abundantly expressed cytokine. Therefore, we investigated whether serum IL-6 levels could be clinically useful parameters of disease activity in rheumatoid arthritis (RA). The serum IL-6 levels were measured by enzyme-linked immunosorbent assay. The IL-6 levels were significantly higher in sera from patients with RA (n = 108, 13.3 +/- 14.0 pg/mL) than in those from the normal controls (n = 10, < 3.13 pg/mL). The IL-6 levels significantly correlated with conventional parameters, such as the Lansbury index, joint score, erythrocyte sedimentation rates, and C-reactive protein. In addition, the serum IL-6 level was useful to determine clinical remission. Advantages of serial measurement of IL-6 were observed in two cases. These findings suggested that the serum IL-6 level may be clinically useful as a sensitive parameter of disease activity in RA.
Abstract: OBJECTIVE: Inflammatory cytokines have been implicated as important mediators of inflammation in rheumatoid arthritis (RA). We investigated whether interleukin 4 (IL-4) and interleukin 10 (IL-10) suppress the production of inflammatory cytokines by freshly prepared adherent rheumatoid synovial cells. METHODS: Adherent synovial cells were obtained from the rheumatoid synovium by collagenase digestion. The levels of IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 in culture supernatants were measured by ELISA. The gene expression of IL-6 and IL-8 were determined by Northern blot analysis. RESULTS: Freshly prepared rheumatoid synovial cells spontaneously produced large amounts of IL-6 and IL-8. However, the amounts of IL-1 beta and TNF-alpha produced were approximately 1000-fold less than those of IL-6 and IL-8. IL-4 alone inhibited the production of IL-1 beta, IL-6, and IL-8 by 32, 35, and 50%, respectively. IL-10 alone was less potent than IL-4 in suppressing these cytokines. Of note, the combination of IL-4 and IL-10 cooperatively exerted potent suppressive effects on the production of IL-1 beta, IL-6, and IL-8 by 74.3, 69, and 77%, respectively. The suppressive effects of the combination of IL-4 and IL-10 on IL-6 and IL-8 were also observed at the levels of mRNA. CONCLUSION: These results suggest that combination of IL-4 and IL-10 may be capable of suppressing the production of inflammatory cytokines at rheumatoid inflammatory joints.
Abstract: An injectable emulsion of 10% tridihomo-gamma-linolenoyl glycerol (DGLA-TG, 96% pure) was prepared. 0.5 ml of the emulsion was injected into tail veins of 6-week-old C3H/HeSlc mice. They were killed 1, 3, 6, 12 and 24 h after the injection. The fatty acid composition of the phospholipid (PL) fraction of plasma, splenocytes and thymocytes was analysed along with that of control mice. DGLA concentrations increased markedly 1 h after the injection in the plasma (from 2.2% to 13.2%) and splenocyte (from 1.1% to 10.1%) PL fractions; they decreased rapidly thereafter. On the other hand, DGLA concentrations in the thymocyte PL fraction did not increase markedly. These data may be useful for planning animal experiments with DGLA emulsions, should these be developed as an experimental drug in the future.
Abstract: 1. We have formulated an infusible emulsion of 10% tridihomo-gamma-linolenoyl-glycerol (94% pure) and investigated the effects of its infusion on leukotriene B4 production. 2. We infused the emulsion into the ear veins of two rabbits in a dose of 0.8g of tridihomo-gamma-linolenoyl-glycerol/kg. Polymorphonuclear leucocytes were obtained from rabbits before the infusion, and 6, 24, 72 and 168 h after. The ionophore-stimulated leukotriene B4 production by polymorphonuclear leucocytes was reduced to about 50% of baseline as early as 6 h after the infusion and remained reduced until 24 h after the infusion. 3. To closely investigate the changes in leukotriene B4 production at 6 h after the infusion, we infused 1.0 g of tridihomo-gamma-linolenoyl-glycerol/kg into the tail veins of eight male Wistar rats. Six hours later, peritoneal macrophages were obtained from four rats for the analysis of ionophore-stimulated leukotriene B4 production. Peritoneal cells and blood samples were also obtained from the remaining rats for fatty acid analysis. Another eight rats were used without prior infusion for baseline determination. 4. The production of leukotriene B4 by macrophages was significantly reduced to 42% of baseline, and the plasma free dihomo-gamma-linolenic acid was markedly increased from 0.26 to 4.82 mol%, in 6 h. 5. We conclude that the infusion of a tridihomo-gamma-linolenoyl-glycerol emulsion is able to decrease leukotriene B4 production and to increase the dihomo-gamma-linolenic acid concentrations in the plasma and peritoneal cells of rats in 6 h.
Abstract: Leukotriene (LT) B4 is a major chemical activator of PMN. Inhibitory effects of oral administration of docosahexaenoic acid (DHA) on LTB4 synthesis by PMN are known. We intravenously infused tridocosahexaenoyl-glycerol (DHA-TG) emulsion into rabbits in three different doses, namely 0.8, 0.4, or 0.2 g DHA/kg, and investigated the changes in LTB4/5 production by ionophore-activated PMN. The averaged LTB4 production by PMN was significantly reduced to 57 and 59% of baseline at 6 h after the infusion of 0.8 and 0.4 g DHA/kg, respectively (P < 0.05), but not after the infusion of 0.2 g DHA/kg or 0.8 g soybean oil/kg. The combined concentrations of both DHA and eicosapentaenoic acid in the PMN phospholipid fraction were significantly increased at 6 h after the infusion of 0.8 or 0.4 g DHA/kg but not after the infusion of 0.2 g DHA/kg or 0.8 g soybean oil/kg. Oral administration of 0.8 g DHA/kg did not increase DHA or eicosapentaenoic acid in the PMN phospholipid fraction and did not decrease LTB4 production by PMN at 6 h after administration. We suggest that the infusion of 0.4-0.8 g DHA/kg might be beneficial to patients who suffer from diseases that are related to the acute elevation of LTB4 production.
Abstract: Highly purified tridihomo-gamma-linolenoyl-glycerol (DGLA-TG) was emulsified with egg yolk lecithin as a 10% (wt/vol) DGLA-TG emulsion. We injected 0.05 or 0.5 mL of the emulsion into mice through the tail vein and investigated its effects on the fatty acid composition of spleen cells and on delayed-type hypersensitivity (DTH) response. At 1 h after the injection, dihomo-gamma-linolenic acid (DGLA) concentrations were increased significantly in the total phospholipid fraction of spleen cells from 1.21 +/- 0.13 mol% to 2.09 +/- 0.74 mol% (P < 0.02) and 7.95 +/- 1.25 mol% (P < 0.001) in the 0.05-mL and 0.5-mL groups, respectively. Mice, which had already been immunized with sheep red blood cells (SRBC), were challenged by the injection of SRBC into the right-hind footpad. Intravenous injection into mice with 0.5 mL of the emulsion immediately before the challenge almost completely suppressed DTH response measured by the swelling of the right-hind footpads 24 h thereafter. This inhibitory effect on the DTH response was significant with as little as 0.05 mL of the emulsion, whereas a soybean oil emulsion was not effective at all. In conclusion, intravenous injection of a DGLA emulsion increased DGLA concentrations in immune cells within 1 h and suppressed the DTH reaction.
Abstract: The effects of injection of n-3 polyunsaturated fatty acids (PUFAs) on the delayed-type hypersensitivity (DTH) response was investigated in mice. Mice were immunized with sheep red blood cells (SRBCs). Six days later 50 microliters of a 20% SRBC suspension was injected into the right hind footpad of each mouse. Just before the challenge of SRBCs, various amount of a trieicosapentaenoyl-glycerol emulsion (10%) was injected through tail veins (5 mice per each dose). Then 24 hr later the dorsoventral thickness of the right hind footpad was measured and compared with that of the left hind footpad. The difference in thickness between both footpads was regarded as the DTH response. The effect of the emulsion on DTH was dose-dependent; the DTH responses (in mm) in the control group (injected with 0.5 ml of a 2.5% glycerol solution through tail veins) and EPA-injected groups (with 5 mg, 10 mg, and 20 mg) were 1.53 +/- 0.16 (mean +/- SEM), 1.09 +/- 0.14, 0.43 +/- 0.07 (P less than 0.005), and 0.36 +/- 0.13 (P less than 0.005), respectively. The DTH response was also depressed by the injection of a tridocosahexaenoyl-glycerol emulsion. Consequently, n-3 PUFA emulsions have clinical implication in DTH-related diseases such as rejection of allografts.
Abstract: The effect of age on eicosapentaenoic acid (20:5 n-3; EPA) incorporation into plasma lipids was investigated in young volunteers (8 males, 19 +/- 1 yr) and middle-aged volunteers (6 males, 53 +/- 7 yr). They were asked to take 5.4 g fish oil per day for one week. The increment in EPA in the cholesteryl ester fraction after the supplementation was significantly greater in the middle-aged group (delta = 1.69%) than in the young group (delta = 0.44%) (p less than 0.05). The food intake analyzed for 3 consecutive days just before the supplementation revealed that the young group took more linoleate (17 vs. 10 g) than the middle-aged group. There was a significant inverse correlation between the increment in EPA in the cholesteryl ester fraction after the supplementation and daily linoleate intake among all the volunteers combined (r = -0.63, p less than 0.02). The higher increment in EPA in cholesteryl ester in the middle-aged group might be due to less intake of linoleate and not due to the difference in age itself.
Abstract: Streptozotocin (45 mg/kg) was intravenously administered to 7-week-old Wistar rats through their tail veins. After 11 days, the rats were divided into two groups. One group was fed a lipid-free diet (90%, w/w) plus lard (8%) and safflower oil (2%) for four weeks (Diet 1 group, n = 12). The other group was fed in the same way, except that safflower oil was replaced by 90% pure eicosapentaenoic acid (EPA) ethyl ester (Diet 2 group, n = 13). Twenty-four-hour urine was collected just before the diets started and during the experiment at 7-day intervals. In the second and third week, the levels of proteinuria were significantly lower in the Diet 2 group than they were in the Diet 1 group. There was no significant difference in the levels of creatinine, urea nitrogen, or lipids in plasma or in body weights between the two groups after four weeks on the diets. Because Diet 2 reduced proteinuria of diabetic rats compared to Diet 1, an EPA-rich diet may retard the development of diabetic nephropathy.
Abstract: Pulse wave velocity (PWV) of the aorta was measured in 55 inhabitants of fishing villages and in 49 inhabitants of farming villages, where people normally eat less fish than in the fishing villages. The PWV was significantly slower (indicating less sclerosis) in the fishing villages than in the farming villages (P less than 0.005). This is consistent with a lower incidence of ischemic heart disease in a coastal area, which includes the fishing villages, than in a mountainous area, including the farming villages. It is reported that a long-term fish-diet slows down sclerotic changes of arteries.
