ABSTRACT
OBJECTIVES: The complement system has been proposed to play a significant role in the regulation of T-cell responses. However, the precise mechanism underlying C4-induced immune tolerance remains to be clarified. We recently reported that monomeric C4b inhibits CXCL10 production from blood cells. The purpose of this study was to verify the active site of monomeric C4b. MATERIALS AND METHODS: We investigated the in vitro effects of a C4b-derived peptide (VPAGSARPVAFSVVPTAAA), named HP2 (highly homologous peptide 2), on the IFN-ß-induced production of CXCL10 in human blood and the in vivo effects of the administration of HP2 on Th1/2 cytokine production in the spleen in mice. We also tested whether the administration of HP2 influences symptoms of experimentally induced ulcerative colitis in mice. RESULTS: HP2 inhibited CXCL10 production in human blood, and the administration of HP2 significantly suppressed the production of Th1 cytokines, such as IL-2, IFN-γ, and TNF-α, in spleen cells isolated from mice. The administration of HP2 in the mice significantly improved the symptoms of colitis, with down-regulation of colitogenic CD4(+)CD45RB(high) T cells and up-regulation of CD4(+)LAP/TGF-ß1(+) T cells. CONCLUSION: The amino acid sequence described above is suggested to be the active site in C4b for the inhibition of Th1 cytokine production. These results should contribute to the development of new drugs suppressing autoimmune responses.
Subject(s)
Complement C4b/chemistry , Cytokines/immunology , Immunosuppressive Agents/pharmacology , Peptides/pharmacology , Adult , Amino Acid Sequence , Animals , Colitis, Ulcerative/drug therapy , Colitis, Ulcerative/immunology , Female , Humans , Immunosuppressive Agents/therapeutic use , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Peptides/therapeutic use , Protein Structure, Tertiary , Spleen/cytology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , Th1 Cells/drug effects , Th1 Cells/immunology , Th17 Cells/drug effects , Th17 Cells/immunology , alpha-DefensinsABSTRACT
Gene expression of transforming growth factor-beta (TGF-beta) is needed to induce expression of transcription factor forkhead box P3 (Foxp3), which is required for the development and function of regulatory T (Treg) cells. The number of circulating Treg cells and the level of Foxp3 expression increase during granulocyte and monocyte apheresis (GMA), a useful therapy for ulcerative colitis. However, the mechanism underlying GMA-induced Foxp3 expression is unknown. We found that the level of TGF-beta mRNA in peripheral blood mononuclear cells (PBMCs) was augmented just after treatment of peripheral blood with a GMA carrier, cellulose acetate beads, in vitro and that Foxp3 expression in PBMCs increased after culturing these cells for 5 days after the treatment. The augmentation of TGF-beta expression was observed in CD3(-) PBMCs but not in CD3(+) T cells. Furthermore, the increase in Foxp3 expression in T cells depended on co-culture with CD3(-) PBMCs. We conclude that cellulose acetate beads have an ability to induce Foxp3 expression in peripheral blood T cells via augmentation of TGF-beta expression in CD3(-) PBMCs.
Subject(s)
Cellulose/analogs & derivatives , Forkhead Transcription Factors/genetics , T-Lymphocytes, Regulatory/metabolism , Blood Component Removal/methods , CD3 Complex , Cellulose/pharmacology , Coculture Techniques , Colitis, Ulcerative/therapy , Granulocytes , Humans , Immunophenotyping , Microspheres , Monocytes , T-Lymphocytes, Regulatory/cytology , Transcriptional Activation/drug effects , Transforming Growth Factor beta/drug effectsABSTRACT
Assessment of spleen size using the ultrasonography has become a standard practice in human. However, the assessment is not established method in experimental animals. To establish the index to assess the spleen size using ultrasonography, we measured the cross-section image of rabbit spleen during endotoxin shock. The image of the cross-section was appeared as triangle, and the height of the triangular image was defined as the spleen index. This spleen index showed strong correlation with the spleen weight. In conclusion, this method is suitable for observation of changes in rabbit spleen size and may reduce the number of rabbit in the longitudinal studies.
Subject(s)
Rabbits/anatomy & histology , Spleen/anatomy & histology , Spleen/diagnostic imaging , Animals , Organ Size , UltrasonographyABSTRACT
Granulocyte and monocyte adsorptive apheresis (GMA) using a column filled with cellulose acetate (CA) beads (carriers) has been associated with a significant clinical efficacy in patients with rheumatoid arthritis and ulcerative colitis. To obtain further understanding on the mechanisms of disease modification by cellulose acetate-carrier-based GMA, in the present study, we investigated the mechanisms of granulocyte and monocyte adhesion to CA beads following exposure of human peripheral blood to the carriers at 37 degrees C for up to 60 min under controlled conditions. Cellulose acetate beads selectively adsorbed granulocytes, monocytes. CD19+ (B cells) and CD56+ (NK cells) lymphocyte subpopulations. The granulocyte and monocyte adsorption was inhibited by heat-inactivated plasma and EDTA, indicating that the adsorption was plasma protein (immunoglobulin, complement) and calcium dependent. Accordingly, granulocyte and monocyte adsorption was markedly enhanced by coating the carriers with IgG. Similarly, C3b was adsorbed onto the CA beads as a marker of complement activation. The results indicated that IgG and active complement fragments mediated leukocyte adhesion to CA beads via the FcgammaR and/or leukocyte complement receptor like CR3. Additionally, CA beads induced loss of expression of TNF receptors on CD16- granulocytes and CD14+ monocytes, but not on CD3+ lymphocytes In conclusion, CA beads might be an appropriate biomaterial for inducing extracorporeal immunomodulation as a treatment for auto-immune diseases which are associated with pathological leukocyte activity.
