Production of autoantibodies against citrullinated antigens/peptides by human B cells.

Autoantibodies against citrullinated protein Ags (ACPA) are associated with the development of rheumatoid arthritis (RA). This immune response against citrullinated protein Ags, which is thought to be facilitated by certain MHC HLA-DR alleles, is highly specific for this disease and has been speculated to be involved in the pathogenesis. We have previously studied cultures of B cells for the production of Abs against HLA Ags. The aim of the current study was to examine the role of B cells in the production of ACPA in patients with RA. Peripheral blood B cells from RA patients and healthy people were cultured with EL4-B5, a murine cell line expressing human CD40L, and with T cell factors to stimulate the in vitro production of Abs by B cells isolated from peripheral blood. ACPA were produced by cultured B cells from RA patients, as determined by reactivity to cyclic citrullinated peptide (CCP). The results showed that 22% of the healthy persons tested also had B cells that could produce ACPA. Patients with HLA-DR alleles carrying the RA-associated shared epitope appeared to have more B cells with autoimmune potential for CCP than those without such HLA alleles (odds ratio 8.1, p = 0.001). In healthy individuals, anti-CCP-producing B cells were also observed more frequently if the RA-associated MHC genes were present (odds ratio 8.0, p = 0.01). Analysis of B cells in cultures may shed light on the interaction of genetic and environmental factors in the development of RA.

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly.

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.

[Study on the metabolism of cartilage matrix by the chondrocytes in osteoarthritic condylar cartilage].

OBJECTIVE: To study the characteristics of cellular metabolism of mandibular condylar chondrocytes in repairing state of osteoarthrosis and investigate its role in the pathogenesis of the disease. METHODS: Temporomandibular joint osteoarthrosis model of rabbits was created by the partial resection of joint disc and confirmed with histological diagnosis. The chondrocytes were harvested from osteoarthritic condylar cartilage in the repairing state and cultured in vitro under the monolayer culture condition. The cellular expression of cartilaginous matrix protein, collagenase and growth factors between the osteoarthritic chondrocytes and the normal controls were measured with RT-PCR technique to outline the basic feature of the osteoarthritic cells. RESULTS: The cultured cells were confirmed as chondrocytes with their ability of expression of collagen type II and Aggrecan. In the reactive repairing state of osteoarthrosis, the chondrocytes showed the imbalance of expression of ECM proteins, and increased expression of collagenase and endogenous growth factors such as IGF-1 and TGF-beta1. CONCLUSIONS: This study found the active anabolism of the chondrocytes within the osteoarthritic condylar cartilage and the imbalance synthesis of cartilage matrix. These repairing attempts by the osteoarthritic chondrocytes may be impossible to restore the primary homeostasis within the condylar cartilage.

The 5'-upstream region of human programmed cell death 5 gene contains a highly active TATA-less promoter that is up-regulated by etoposide.

The PDCD5 (programmed cell death 5), a novel apoptosis related gene, is functionally associated with cell apoptosis, exhibits a ubiquitous expression pattern and is up-regulated in some types of tumor cells undergoing apoptosis. To study the transcriptional regulation of the PDCD5 gene, we have cloned 1.1 kb of its 5'-upstream region. The DNA sequencing analysis revealed a major transcriptional start site at 72 base pairs in front of the ATG translational start codon. The upstream of the transcriptional start site lacks a canonical TATA box and CAAT box. Transient transfection and luciferase assay demonstrate that this region presents extremely strong promoter activity. The 5'-deleted sequences fused to a luciferase reporter gene demonstrated that the -555/-383 region from the transcription start site is crucial for transcriptional regulation, and the luciferase reporter gene's expression significantly increased in the early stage of cell apoptosis induced by etoposide. These results imply that the PDCD5 gene may be a target gene under the control of some important apoptosis-related transcriptional factors during the cell apoptosis.

Last intron of the chemokine-like factor gene contains a putative promoter for the downstream CKLF super family member 1 gene.

The genes for chemokine-like factor (CKLF) and four chemokine-like factor super family members (CKLFSF1-4) are tightly linked on chromosome 16, with only 325 bp separating CKLF and CKLFSF1. We used Northern blotting and RT-PCR to show that these two genes are expressed independently of one another. We then used a novel computational promoter prediction method based on the interaction among transcription factor binding sites (TFBSs) to identify a putative promoter region for the CKLFSF1 gene. Our method predicted a promoter region in the last intron of the upstream gene, CKLF. We PCR amplified the predicted promoter region and used a luciferase assay to show that the region was able to drive the luciferase gene. DNA decoy experiments indicated that 214 bp fragment neighboring the TATA box markedly inhibited CKLFSF1 gene expression. Sequence analysis of the region revealed a typical TATA box (TATATAA) and multiple potential transcription factor binding sites, providing further evidence for this being a functional promoter for CKLFSF1. This work provides the first evidence of a promoter from one gene located in an intron of another.

Molecular cloning and characterization of four isoforms of mCKLF, mouse homologues of human chemokine-like factor.

Chemokine-like factor1 (CKLF1), and its three isoforms (CKLF2, 3 and 4), are recently identified human cytokines. CKLF1 is a potent chemoattractant for human leukocytes and can stimulate inflammation and the regeneration of murine skeletal muscle. CKLF2 can promote proliferation and differentiation of C2C12 muscle cells directly by inducing expression of myogenin and activating transcription factors. In the present study, we cloned CKLF murine homologues, and based on their biological and structural features, named them murine chemokine-like factor 2, 4, 5 and 6 (mCKLF2, 4, 5 and 6). mCKLF2, 4, 5 and 6 encode 152, 120, 122 and 86 amino-acid proteins, respectively. mCKLFs map to mouse chromosome 8 and have high sequence similarity to human CKLFs. Compared to human CKLFs, which have a CC motif in the C-terminal region, mCKLF2 and 4 contain a CX3C motif. Using a PCR-based approach, it appeared mCKLF2 and 5 mRNA were highly expressed in adult testis, while mCKLF4 mRNA was detected only in differentiated C2C12 cells, a pattern different from human CKLFs. Conditioned media from COS-7 cells transfected with mCKLF2 and 4 was chemotactic for mouse neutrophils, macrophages and lymphocytes. Our results show that mCKLF2, 4, 5 and 6 are four splicing variants which are homologues of human CKLFs and murine CKLFs possess distinct features compared to their human counterparts.

Molecular cloning and characterization of rat chemokine-like factor 1 and 2.

Chemokine-like factor 1(CKLF1) is a newly cloned cytokine with three RNA splicing isoforms. It has chemotactic activities on leukocytes and plays an important role in skeletal muscle regeneration. Here we have isolated two rat homologues of human chemokine-like factors by expressed sequence tag assembly, which are designated as rat chemokine-like factor 1 and 2 (rat CKLF1, CKLF2). The full-length cDNAs of rat CKLF1 and -2 contain 523 and 682 nucleotides and the open reading frames encoding 98 and 151 amino acids, respectively. Rat CKLF1 and -2 share about 54.1 and 59.6% homologies with human CKLF1 and -2 at the amino acid level; both rat CKLF1 and -2 contain a CX3C motif at their C-terminal regions while human CKLFs have a CC motif at the same regions. Rat CKLFs are highly expressed in testis, while human CKLFs have a broad expression spectrum across multiple tissues. Recombinant rat CKLF1 can be secreted into the cell culture supernatants and has chemotactic effects on neutrophils, macrophages and lymphocytes, which is similar to human CKLF1, while recombinant rat CKLF2 has weaker chemotactic effects on these cells. These findings show that rat CKLFs have similar bioactivity with human CKLFs, although they are different in tissue distribution and contain different characteristic motifs.

[A novel cis-acting enhancer element between CKLF and CKLFSF1 genes].

AIM: To explore the roles of the CKLF gene and CKLFSF1 gene sequence (CCS) in transcriptional regulation. METHODS: The target gene fragment was amplified by PCR and then inserted into pGL3-basic and pGL3-SV40 containing luciferase reporter vector gene to construct pGL3-basic-CCS and pGL3-SV40-CCS. Using liposome-mediated method, four recombinant plasmids were respectively transfected into Hela cells. Transient expression was analyzed. RESULTS: The luciferase assay indicated that the no luciferase activity was detected in Hela cells transtected with pGL3-basic and pGL3-basic-CCS. However, the luciferase activity was doubled when pGL3-SV40-CCS was transfected into Hela cells. CONCLUTION: The CCS has no promoter activity, whereas some important cis-acting enhancer elements which modulate its downstream gene expression may exist within this sequence.

Overexpression of chemokine-like factor 2 promotes the proliferation and survival of C2C12 skeletal muscle cells.

Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells. It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of CKLF2, which is the largest isoform of CKLFs, C2C12 murine myoblasts were stably transfected with human CKLF2 eukaryotic expression vector. Compared with control vector transfected C2C12 cells, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [(3)H]TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. Finally, DNA synthesis and myotube formation could also be promoted by growing C2C12 cells in conditioned media from CKLF2-transfected cells. These findings strongly suggest a role for human CKLF2 in regulation of skeletal muscle myogenesis.

[Calcitonin gene-related peptide induces chemokine interleukine-8 synthesis in human monocytes].

OBJECTIVE: To study the effect of calcitonin gene-related peptide (CGRP) on the function of immune cells. METHODS: Human monocytes were cultured with calcitonin gene-related peptide in vitro and activated with lipopolysaccharide (LPS). The IL-8 level in the supernatant was measured with ELISA and the IL-8 mRNA expression in monocytes was observed by reverse transcription polymerase chain reaction (RT-PCR). The chemotactic activity of monocytes to neutrophils and lymphocytes was analyzed with micro-chemotacxis chamber. Chemotactic index (CI) was calculated by the formula: number of monocytes migrating to the underside of membrane in the LPS + CGRP group/number of monocytes migrating to the underside of membrane in the control group. CGRP receptor antagonist CGRP8 - 37 was added into the culture to study the effect of CGRP. Blank control and cultures of monocytes with LPS or with CGRP only were used as controls. RESULTS: The level of IL-8 protein in the supernatant of the LPS + CGRP group was 1 120 pg/ml +/- 14.80 pg/ml, significantly higher than those in other groups (670 pg/ml +/- 15.10 pg/ml in LPS + CGRP + CGRP8 - 37 group). The expression of IL-8 mRNA in the LPS + CGRP group was the highest (IL-8/beta-actin = 1.845 +/- 0.587), IL-8/beta-actin in the LPS + CGRP + CGRP8 - 37 group was1.339 +/- 0.434. The chemotactic activities of the monocytes to neutrophils and lymphocytes were enhanced in the LPS + CGRP group (CI = 3.78 +/- 0.08 to neutrophils and CI = 3.4 +/- 0.27 to lymphocytes). The CI values were 1.15 +/- 0.31 and 1.21 +/- 0.06 respectively in the LPS + CGRP + CGRP 8 - 37 group. CONCLUSION: CGRP in the peripheral nerve ending induces monocytes to synthetize and secret chemotactic factor IL-8 and enhance the chemotactic activity of monocytes, thus promoting the directional migration and aggregation of neutrophils and lymphocytes to foci of inflammation.