Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor beta-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(L-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.
To gain insight into the nature of the immune response with respect to accumulation and composition of the T-cell receptor (TCR) repertoire in synovial tissue in rheumatoid arthritis (RA), we have determined the nucleotide sequence of TCRBV regions transcribed by T lymphocytes derived from synovial tissue. Synovial tissue was obtained by needle biopsies from three different sites of the same joint in two early RA patients. We found that the TCRBV region repertoire among synovial tissue-infiltrating mononuclear cells was heterogeneous when the different biopsies taken from each patient were compared. However, DNA sequence analysis of TCRBV rearrangements of synovial T lymphocytes showed conserved amino acid usage profiles in the CDR3 domains of different TCRBV regions, which exhibited an individual specific character. These CDR3 motifs were not present in paired samples of peripheral blood. The existence of homologous CDR3 amino acid profiles within the TCRBV regions derived from synovial tissue is indicative of an antigen-driven immune response.
Background and objectiveRheumatoid Arthritis (RA) is a heterogeneous disease affecting approximately 1–2% of the European population. Development of RA is affected by both genetic and environmental triggers. In the present study we hypothesised that these environmental triggers might induce changes in the epigenome of immune cells. A concept that describes such a mechanism is known as ‘trained immunity’, which shows that isolated monocytes previously exposed to micro-organisms display elevated H3K4me3 on the promotor regions of immune genes resulting in enhanced RNA- and protein levels of TNFa and IL6 upon re-stimulation with inflammatory triggers. The phenomenon that innate cells triggered by past insults more readily react to new non-specific stimuli could contribute to the onset of auto-immunity. We wished to test whether the innate immune system of RA patients displays signs of “trained immunity” by analysing immune responsiveness and epigenetic changes of CD14+ monocytes isolated from both untreated RA patients and age-matched healthy controls.MethodsCD14+ monocytes were isolated from both untreated RA patients and age-matched healthy controls. Secreted TNFa and IL6 levels were measured using ELISA in the supernatant of naïve and LPS-stimulated monocytes from both RA patients and non-RA controls. RNA levels of TNFa and IL6 in naïve and LPS-stimulated monocytes from both groups were examined using RT-qPCR.ResultsBoth RNA and protein levels of TNFa and IL6 were low in unstimulated monocytes isolated from RA patients and healthy controls but no large differences were observed. After LPS-stimulation, the relative RNA levels (expressed in fold change relative to household gene) increased but were similar between patients and controls for TNFa (RA: 1.7 ± 0,4, non-RA: 1.8 ± 0.9) and IL6 (RA: 2.9 ± 1,3, non-RA: 3.3 ± 2.6). Concordantly, no large differences were detected for secreted cytokine levels, although secreted TNFa (RA: 4.3ng/ml ± 2.9, non-RA: 2.8ng/ml ± 2.6) and IL6 (RA: 67.2mg/ml ± 14.6, non-RA: 50.8mg/ml ± 17.3) levels seem slightly elevated in RA monocytes.ConclusionsIn general, we could not detect large differences on RNA level or protein level of TNFa and IL6 between RA and non-RA patients. Therefore, we did not find evidence that the concept of trained immunity may contribute to the development of RA.
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