Synovial vascular patterns and angiogenic factors expression in synovial tissue and serum of patients with rheumatoid arthritis.

OBJECTIVE: To determine whether subgroups of rheumatoid arthritis (RA) patients classified according to their synovial vascular pattern have a different expression of angiogenic mediators or exhibit distinct clinical or biological characteristics. METHODS: Arthroscopies were performed in 27 patients with RA and synovial samples were obtained. Vascular morphology was classified in three patterns: straight (S), tortuous (T) and mixed (M). Immunostaining was performed with anti-vascular endothelial growth factor (anti-VEGF), anti-vascular endothelial growth factor receptor (VEGFR)-1, anti-VEGFR-2, anti-IL-8 and anti-TGF-beta, and measured by digital image analysis. Serum levels of VEGF, TGF-beta and IL-8, and clinical, radiographic and serological data were also analysed. RESULTS: Eleven (41%) patients had the S pattern, nine (33%) the M pattern and seven (26%) the T pattern. The S and M groups had a higher prevalence of rheumatoid factor positivity and erosive disease, and higher levels of markers of systemic inflammation compared with the T group. Synovial expression of VEGF was higher in the S and T groups compared with the M group, whereas TGF-beta was higher in the T compared with the S and M groups. Distinct synovial distribution of VEGF and TGF-beta between groups was also observed. CONCLUSIONS: This preliminary study suggests that RA patients with the S and M patterns share different clinical, biological and serological characteristics compared with those with the T pattern, which may constitute a group with less severe disease. Differences in the intensity and distribution of synovial expression of VEGF and TGF-beta observed between groups could have pathophysiological relevance. However, larger, prospective multicentre studies would be need to determine the clinical relevance of vascular patterns in RA.

Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation.

Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes.

Generation of MHC class I peptide antigens by protein processing in the secretory route by furin.

Cytosolic degradation of endogenously synthesized proteins by the proteasome and translocation of processed peptides to the endoplasmic reticulum by the transporters associated with antigen presentation constitutes the classical route for antigen presentation by MHC class I proteins. We have previously defined an alternative pathway in the secretory route involving proteolytic maturation of precursor proproteins for chimeric hepatitis B virus secretory core protein HBe containing a class I epitope at its carboxy-terminus. We extend those results by demonstrating that intracellular delivery of the trans-Golgi network protease furin increases both proteolytic maturation and antigen presentation of the chimeric HBe proteins. An additional class I epitope from the HIV envelope gp160 protein was inserted into this COOH-terminal region of two different chimeric HBe proteins. This epitope was also presented to CTL in a transporter-independent manner involving furin, and protein maturation and antigen presentation were also enhanced by furin over-expression. Presentation of this second epitope was restricted by a different class I allele, thus suggesting that antigen presentation by this new pathway may apply to any antigenic epitope and class I molecule. These results define the furin proteolytic maturation pathway of HBe in the secretory route as a general antigen processing route for MHC class I presentation.

Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin.

Classical antigen presentation by major histocompatibility complex class I molecules involves cytosolic processing of endogenously synthesized antigens by proteasomes and translocation of processed peptides into the endoplasmic reticulum (ER) by transporters associated with antigen presentation (TAP). Alternative pathways for processing of endogenous antigens, generally involving the ER, have been suggested but not fully proved. We analyzed the potential for class I presentation of proteolytic maturation of secretory antigens in the exocytic pathway. We found that hepatitis B (HB) virus secretory core protein HBe can efficiently deliver COOH-terminally located antigenic peptides for endogenous class I loading in the absence of TAP. Antigen presentation to specific cytotoxic T lymphocytes correlates with protein maturation at the COOH terminus, since modification of maturation and transport of HBe through the secretory pathway alters antigen presentation. Both maturation and a necessary processing step occur in the Golgi or post-Golgi compartment. Antigen presentation is independent of proteasome activity, but inhibitors of the trans-Golgi network resident protease furin inhibit both HBe maturation and antigen presentation. These results define a new antigen processing pathway located in the secretory route, with a central role for proteolytic maturation mediated by the subtilisin protease family member furin as an efficient source for antigen presentation.

Expression of different mitogen-regulated protein/proliferin mRNAs in Ehrlich carcinoma cells.

Results from in vivo and from serum-free primary cultures of Ehrlich cells suggest that the expression of mitogen-regulated protein/proliferin (MRP/PLF) mRNAs is not essential for proliferation of this murine tumor. Two sizes for MRP/PRL-related open reading frames (ORFs) have been detected by reverse transcription/PCR amplification. They are almost identical to that reported for PLF-1; but 20% of the amplified cDNA included a shorter ORF, which lacks the entire sequence corresponding to that of the exon 3 of the mrp/plf genes. Ehrlich carcinoma may represent a good model to study regulation of expression and physiological roles of MRP/PLFs in vivo.