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A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells.

ABSTRACT: Mild digestion of 125I-labelled human proteoglycan aggregates with trypsin or stromelysin produced specific peptides that were taken up rapidly by THP-1 monocytes. SDS/PAGE of undigested aggregate showed that the three components of molecular mass 48, 44 and 41 kDa, corresponding to isoforms of link protein originally present, had been converted into a single component of 41 kDa by trypsin treatment, and that fragments of 6-12 kDa were present in fractions containing the high-uptake peptide. Separate proteolysis of isolated proteoglycan monomer and link protein confirmed that the specific high-uptake fragment was derived from link protein. Uptake of the link fragment was rapid, reaching a maximum after 5 min, and specific, since it was blocked by metabolic or serine proteinase inhibitors and at 4 degrees C. After uptake the cleaved fragment was processed further, with 50% of the radiolabel being released as degraded peptides within 5 min. In contrast, accumulation of whole aggregate reached a maximum after 45 min and only 50% had been released after 2 h. Uptake of aggregate was less affected by inhibitors or at low temperature, suggesting that a separate mechanism existed for its turnover. The aggregate was transported to lysosomes after uptake, although the link fragment did not sediment with either lysosomes or plasma membranes, suggesting that it was present in the cytoplasm or in very labile vesicles. However, the mode of handling of the peptide by the cells remains unclear. The link fragment was taken up by several different monocytic and B cell lines, but not by mouse fibroblasts or peritoneal macrophages. These data suggest that a surface serine proteinase on monocytes and B cells enables them to process and take up a fragment of link protein derived by extracellular proteolysis.

SUBMITTER: Martin H

PROVIDER: S-EPMC1130507 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Project description:Purpose Dysfunction of the blood-brain barrier (BBB) and albumin extravasation have been suggested to play a role in the etiology of human epilepsy. In this context, dysfunction of glial cells attracts increasing attention. Our study was aimed to analyze in the hippocampus (1) which cell types internalize albumin injected into the lateral ventricle in vivo, (2) whether internalization into astrocytes impacts their coupling and expression of connexin 43 (Cx43), and (3) whether expression of Kir4.1, the predominating astrocytic K(+) channel subunit, is altered by albumin.Methods The patch-clamp method was combined with single cell tracer filling to investigate electrophysiologic properties and gap junction coupling (GJC). For cell identification, mice with cell type-specific expression of a fluorescent protein (NG2kiEYFP mice) and immunohistochemistry were employed. Semiquantitative real time polymerase chain reaction (RT-PCR) allowed analysis of Kir4.1 and Cx43 transcript levels.Key findings We show that fluorescently labeled albumin is taken up by astrocytes, NG2 cells, and neurons, with NG2 cells standing out in terms of the quantity of uptake. Within 5 days postinjection (dpi), intracellular albumin accumulation was largely reduced suggesting rapid degradation. Electrophysiologic analysis of astrocytes and NG2 cells revealed no changes in their membrane properties at either time point. However, astrocytic GJC was significantly decreased at 1 dpi but returned to control levels within 5 dpi. We found no changes in hippocampal Cx43 transcript expression, suggesting that other mechanisms account for the observed changes in coupling. Kir4.1 mRNA was regulated oppositely in the CA1 stratum radiatum, with a strong increase at 1 dpi followed by a decrease at 5 dpi.Significance The present study demonstrates that extravasal albumin in the hippocampus induces rapid changes of astrocyte function, which can be expected to impair ion and transmitter homeostasis and contribute to hyperactivity and epileptogenesis. Therefore, astrocytes may represent alternative targets for antiepileptic therapeutic approaches.

Project description:BackgroundThe pathology of juvenile dermatomyositis (JDM) is characterized by prominent vessel wall and perivascular inflammation. This feature of the disease has remained unexplained and under-investigated. We have hypothesized that plasma exosomes, which play an important role in inter-cellular communication, may play a role in the vascular injury associated with JDM.ObjectiveTo characterize the circulating exosomes of children with JDM and determine whether the small RNA cargoes within those exosomes are capable of altering transcriptional programs within endothelial cells.Design/methodsWe purified exosomes from plasma samples of children with active, untreated JDM (n = 6) and healthy controls (n = 9). We characterized the small RNA cargoes in JDM and control exosomes by RNA sequencing using the Illumina HiSeq 2500 platform. We then incubated isolated exosomes from healthy controls and children with JDM with cultured human aortic endothelial cells (HAEC) for 24 h. Fluorescence microscopy was used to confirm that both control and JDM exosomes were taken up by HAEC. RNA was then purified from HAEC that had been incubated with either control or JDM exosomes and sequenced on the Illumina platform. Differential expression of mRNAs from HAEC incubated with control or JDM exosomes was ascertained using standard computational methods. Finally, we assessed the degree to which differential gene expression in HAEC could be attributed to the different small RNA cargoes in JDM vs control exosomes using conventional and novel analytic methods.ResultsWe identified 10 small RNA molecules that showed differential abundance when we compared JDM and healthy control exosomes. Fluorescence microscopy of labeled exosomes confirmed that both JDM and control exosomes were taken up by HAEC. Differential gene expression analysis revealed 59 genes that showed differential expression between HAEC incubated with JDM exosomes vs HAEC incubated with exosomes from controls. Statistical analysis of gene expression data demonstrated that multiple miRNAs exerted transcriptional control on multiple genes with HAEC.ConclusionsPlasma exosomes from children with active, untreated JDM are taken up by HAEC and are associated with alterations in gene expression in those cells. These findings provide new insight into potential mechanisms leading to the targeting of vascular tissue by the immune system in JDM.

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A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells.

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