The cerebral hemorrhage-producing cystatin C variant (L68Q) in extracellular fluids.

A variant of the normal extracellular cysteine protease inhibitor cystatin C (L68Q-cystatin C), is the amyloid precursor in hereditary cystatin C amyloid angiopathy (HCCAA). It has been suggested that the mutation causes cellular entrapment of L68Q-cystatin C in vivo and that the variant protein is not secreted to extracellular fluids. In order to test this hypothesis, we used matrix-assisted laser desorption ionization time-of-flight mass spectrometry in an effort to demonstrate the presence of L68Q- along with wildtype cystatin C in plasma and cerebrospinal fluid (CSF) of HCCAA-patients. Plasma from all five investigated HCCAA-patients contained both L68Q- and wildtype cystatin C. The presence of approximately equal amounts of cystatin C dimers and monomers was demonstrated in plasma from HCCAA-patients, whereas only monomers could be found in normal plasma. L68Q-wildtype-cystatin C heterodimers seem to be present in the dimeric cystatin C population. CSF from six HCCAA-patients also contained cystatin C-dimers and monomers, but the dimeric fraction was minute. CSF from control patients did not contain dimeric cystatin C. These results suggest that the milieu of L68Q-cystatin C is important for its stability and dimerization status and that certain milieus might hinder its further development into oligomers, amyloid fibrils and other precipitating aggregates.

Nucleotide sequences specific for nonnominal immunoglobulin allotypes in rheumatoid arthritis patients and in normal individuals and their expression in synovial tissue of rheumatoid arthritis patients.

The production of antibodies against nonnominal immunoglobulin allotypes in rheumatoid arthritis (RA) patients suggests that the immune system of these patients has been exposed to such foreign allotypes. The presence of nonnominal allotypes is, however, a genetic enigma. We searched for nucleotide sequences specific for nonnominal G3mg and G3mb copies in individuals homozygous for these alleles. Using a sensitive and specific nested polymerase chain reaction (PCR) method with genomic DNA from blood of 18 RA patients and 5 normal controls, we found G3mg sequences in 18 of 18 tested G3mb homozygous persons. The allele specificity of the PCR fragments was confirmed by sequencing and RFLP analysis. The PCR products contained genomic nonspliced parts of the nonnominal sequences. An analysis of cDNA from inflammatory tissue of 5 RA patients detected nonnominal G3mb sequences in 1 of 3 tested G3mg homozygotes and G3mg sequences in 2 of 2 tested G3mb homozygotes. The cDNA-derived PCR products contained sequences from normally spliced nonnominal Ig fragments. The results also showed that the nonnominal Ig sequences were present in very low copy numbers, lower than the Mendelian 1-2 copies per cell. The origin of such a low copy number of Ig gene fragments may be explained by a virus-mediated capture and transfer mechanism of Ig gene fragments generated by the normal Ig switch-associated gene excision process.

Intracellular accumulation of the amyloidogenic L68Q variant of human cystatin C in NIH/3T3 cells.

AIM: To study the cellular transport of L68Q cystatin C, the cystatin variant causing amyloidosis and brain haemorrhage in patients suffering from hereditary cystatin C amyloid angiopathy (HCCAA). METHODS: Expression vectors for wild-type and L68Q cystatin C were constructed and used to transfect mouse NIH/3T3 cells. Stable cell clones were isolated after cotransfection with pSV2neo. Clones expressing human wild-type and L68Q cystatin C were compared with respect to secreted cystatin C by enzyme linked immunosorbent assay (ELISA), and for intracellular cystatin C by western blotting and immunofluorescence cytochemistry. Colocalisation studies in cells were performed by double staining with antibodies against human cystatin C and marker proteins for lysosomes, the Golgi apparatus, or the endoplasmic reticulum, and evaluated by confocal microscopy. RESULTS: Concentrations of human cystatin C secreted from transfected NIH/3T3 cells were similar to those secreted from human cells in culture. In general, clones expressing the gene encoding L68Q cystatin C secreted slightly lower amounts of the protein than clones expressing wild-type human cystatin C. Both immunofluorescence cytochemistry and western blotting experiments showed an increased accumulation of cystatin C in cells expressing the gene encoding L68Q cystatin C compared with cells expressing the gene for the wild-type protein. The intracellularly accumulating L68Q cystatin C was insoluble and located mainly in the endoplasmic reticulum. CONCLUSIONS: The cellular transport of human cystatin C is impeded by the pathogenic amino acid substitution Leu68-->Gln. The resulting intracellular accumulation and increased localised concentration of L68Q cystatin C might be an important event in the molecular pathophysiology of amyloid formation and brain haemorrhage in patients with HCCAA.

Immunoregulatory cytokines modify Escherichia coli induced uroepithelial cell IL-6 and IL-8 responses.

This study analysed the effects of immunoregulatory cytokines on uroepithelial cell cytokine responses. The A-498 human kidney cell line was treated with the interleukins IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, interferon gamma (IFN-alpha) and transforming growth factor beta (TGF-beta 1). Secreted IL-6 and IL-8 were quantitated by enzyme-linked immunoabsorbent assay (ELISA) and bioassay; IL-6 and IL-8 mRNA species were quantitated by reverse transcriptase polymerase chain reaction (RT-PCR). IL-4, IL-13, IFN-gamma, and TGF-beta 1, but not IL-2, IL-5, IL-10 or IL-12, stimulated IL-6 secretion. At high concentrations, IL-4 and IL-13 stimulated low levels of IL-8 secretion. Immunoregulatory cytokines were analysed for their ability to modify the A-498 cells' IL-6 and IL-8 secretion in response to Escherichia coli. IL-5, IL-12, IL-13 and TGF-beta 1 additively enhanced the bacterially induced IL-6 secretion, but they did not affect IL-8 secretion. The strongest affects on uroepithelial cell IL-6 and IL-8 responses in the presence of bacteria were observed in conjunction with IL-4 and IFN-alpha. IL-4 induced IL-6 production in synergy with E. coli. IFN-alpha both enhanced and inhibited IL-6 and IL-8 responses in combination with E. coli, depending on the order of stimulant addition. The results demonstrate that immunoregulatory cytokines can modify the uroepithelial cell responses to bacteria in vitro. In this way, T cells may regulate the cytokine responses of uroepithelial and possibly other mucosal epithelial cells in vivo.

Promoter-mediated, dexamethasone-induced increase in cystatin C production by HeLa cells.

Cystatin C, an efficient inhibitor of cysteine proteinases, is present in all investigated human extracellular fluids. Dexamethasone caused a significant and dose-dependent increase in the cystatin C secretion of cultivated HeLa cells up to a maximal increase of 80% at 10(-6) mol l-1 dexamethasone. Increased production of cystatin C was also observed at lower concentrations, suggesting that glucocorticoids might play a physiological role in the production of cystatin C. The effect of dexamethasone on the cystatin C gene expression was also studied in a transient transfection expression system using chimeric plasmid constructs of the cystatin C gene promoter (positions -2 to -1084) coupled to the structural gene for human growth hormone (hGH). In this system, a small, but statistically significant, increase in hGH secretion was also observed upon dexamethasone treatment, suggesting that the glucocorticoid-induced increase in secretion of cystatin C is due to a promoter-mediated increase in transcription of the cystatin C gene.

On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy.

The pathogenesis of the deposition of a variant cystatin C as amyloid in hereditary cystatin C amyloid angiopathy (HCCAA) is not known. To address this question the synthesis and secretion of cystatin C in cultured monocytes from 9 carriers of the mutated cystatin C gene (5 symptomatic and 4 asymptomatic) was examined. The quantity of cystatin C in cells and supernatants was determined by the ELISA method, Western blots were done and selected samples immunostained for cystatin C. Monocytes from individuals carrying the gene defect synthesized cystatin C that was apparently not truncated, a form found in the cerebral amyloid deposits in HCCAA, but showed a distinctly lower rate of cystatin C synthesis than monocytes from healthy controls. The main difference was that the quantity of cystatin C was significantly lower in the supernatants in monocyte cultures from carriers of the gene defect than from healthy controls, possibly due to a partial block in its secretion. This abnormal processing of the cystatin C could explain the low cerebrospinal fluid levels of cystatin C in HCCAA and might be a part of the pathogenetic pathway of amyloid deposition. Furthermore it could, through a lower extracellular concentration of this inhibitor of cysteine proteinases, contribute to destruction of the amyloidotic blood vessels, leading to the most serious clinical manifestation in HCCAA, intracerebral hemorrhage.