Objective.: To investigate possible correlations between fingertip blood perfusion (FBP) status, assessed by laser Doppler flowmetry (LDF), and morphological microvascular abnormalities, detected by nailfold videocapillaroscopy (NVC), in patients with systemic sclerosis (SSc). The effects on FBP of intravenous (IV) treatment with the prostacyclin analog iloprost were also investigated.Methods.: Thirty-four consecutive patients with SSc and 16 healthy subjects were evaluated. LDF was performed by analyzing blood perfusion at the fingertips in both hands. Patients with SSc were distributed into the appropriate NVC pattern of microangiopathy (early, active, and late). Iloprost was administered to inpatients with SSc by 24-hour IV infusion for 7 consecutive days (4 μg/h).Results.: FBP was significantly lower in patients with SSc (p < 0.05) compared to controls. Heating of the LDF probe at 36°C induced a significant increase of FBP in all subjects (p < 0.001), but the slope of variation was significantly lower in patients with SSc compared to controls (p < 0.05). Patients with SSc showing the late NVC pattern of microangiopathy had significantly lower FBP than patients with the active and early NVC patterns (p < 0.05). A negative correlation was observed between FBP and NVC rating of the microvascular damage (p < 0.05). After iloprost treatment, a significant increase of FBP was observed in patients with SSc (p < 0.05).Conclusion.: Patients with SSc show a decreased FBP partially reversible by local skin heating. The FBP correlated negatively with the extent of nailfold microvascular damage, and IV iloprost treatment increased the FBP.

Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ET_A and ET_B). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ET_A/BRA, bosentan 10^-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ET_A/BRA treatment in both cultured cell types. ET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ET_A/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.

Objective. We evaluated endothelin (ET)-1 plasma levels and some clinical measures in patients with primary Raynaud’s phenomenon (PRP), and in patients with systemic sclerosis (SSc) and secondary RP (SRP), in the latter according to their different nailfold videocapillaroscopy (NVC) patterns of microangiopathy (early, active, and late).

Introduction: Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested. Methods: All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 μg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) α, IL-1β, transforming growth factor (TGF) β) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed. Results: Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 μg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFα, IL-1β and TGFβ). Data were confirmed by WB and RT-PCR analysis. Conclusions: Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNFα, IL1-β and TGFβ. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.