11/7/2011. Nothing to disclose. Cellular origin of fibrosis. 1. Fibroblasts. 2. Epithelial Mesenchymal Transformation
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1 /7/ T cell dependent regulation of fibrogenesis Financial disclosure Matthias Mack University Hospital Regensburg, Germany Nothing to disclose Cellular origin of fibrosis Acitvation of fibroblasts EMT Fibrocytes. Fibroblasts. Epithelial Mesenchymal Transformation fibroblasts become activated by various factors (e.g. cytokines, TGFb / activin A, ANGII, FGF, PDGF, fibronectin, glucose, mechanical stress) activated fibroblasts (myofibroblasts) express asma, ecto5 nucleotidase, vimentin factors: TGFb EGF, IGFII, FGF MMP factors: BMP7 EMTfibroblasts express: asma (function?) and FSP (also expressed by fibroblasts and leukocytes) EMT has been described in patients: Diab. nephropathy, SLE, IgAN, chron. rejection Kalluri, Zeisberg, Strutz, Neilson and others
2 /7/. Production of collagen by hematopoietic cells fibrocytes What are fibroctyes? First description by R. Bucala: Bucala, R., Spiegel, L. A., Chesney, J., Hogan, M., and Cerami, A. (99). Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med, 7. Fibrocyte precursors Migration Differentiation into fibrocytes Hematopoietic cells present in the peripheral blood and bone marrow, that produce collagen type I after culture about.5 % of total peripheral blood leukocytes Collagen I, CD5 (panleukocyte marker), morphology Detection of blood borne fibrocytes / fibroblasts in patients with chronic allograft nephropathy at mo after Tx Bone marrowderived fibrocytes in pulmonary fibrosis Lung tissue: bleomycin Lung tissue: saline female kidney into male recipient male kidney into female recipient Blue: YChrom. Red: asma (CD5) N Engl J Med ;5:97 Hashimoto et al. JCI Experimental data Precursor cells of fibrocytes In vitro analysis? precursor cells of fibrocytes positive and negative factors for development of fibrocytes _ In vivo analysis detection and modulation of fibrocytes in UUOfibrosis fibrocytes Niedermeier et al. PNAS 9
3 Collagen / GAPDH (mrna) /7/ Development of fibrocytes in culture Development of fibrocytes in culture Fresh blood Fresh spleen Fibrocyte culture 5 d day day 7 day R R R R R R FSC FSC FSC gate gate R d Culture of total splenocytes rabbit IgG α collagen I CD5 CD5 What are the precursor cells of fibrocytes? What are the precursor cells of fibrocytes? CDb CD5 Control CDb µm SSC SSC FSC CDc CD5 FSC Summary Influence of T cells on differentiation of into fibrocytes Gr _ CD T cells acd CyA fibrocytes CD T cells acd SN Niedermeier et al. PNAS 9
4 fibrocytes / hpf SSC fibrocytes / hpf Collagen fibrocytes / hpf /7/ Positve factors for differentiation of into fibrocytes Positve factors for differentiation of into fibrocytes medium CD medium CD CDb CD/ FLH FLH Control FLH CD5 FLH FSC Positve factors for differentiation of into fibrocytes Positve factors for differentiation of into fibrocytes ELISA for intracellular collagen ELISA for secreted collagen Collagen I mrna CDb medium CDb CDb CDb CDb CDb medium medium 95 C atgfb CDb atgfb Positve factors for differentiation of into fibrocytes Summary Gr nonact. CD TZellen act. CD TZellen CNI IL, TGFbeta _ fibrocytes Niedermeier et al. PNAS 9
5 fibrocytes / hpf fibrocytes / hpf Collagen ELISA II (OD) Collagen /7/ Negative factors for differentiation of into fibrocytes Negative factors for differentiation of into fibrocytes medium SN CDb medium FLH CDb SN 5 FLH CDb CDb medium CDb SN CDb FLH CD5 Negative factors for differentiation of into fibrocytes Negative factors for differentiation of into fibrocytes SN αil αil αα αifnγ αil,, Medium IL IL IL IFNg IL IL IFNg IL IFNg IL IL IFNg Summary Experimental data nonact. CD TZellen Gr In vivo analysis unilateral ureteral obstruction act. CD TZellen CNI _ act. CD TZellen IL, TGFbeta IL, a, IFNg, IL fibrocytes day : UUO day 7 : analysis Niedermeier et al. PNAS 9 5
6 Rabbit IgG Collagen I Rabbit IgG acollagen I CDb, CD5, Col I (recovered per kidney) CDb CDb CDb Gr per µl blood recovered per kidney recovered per kidney /7/ Detection of fibrocytes in unilateral ureteral obstruction in mice Collagen I mrna in purified CD5 cells from UUO kidneys UUO kidney contralat. kidney UUO kidney FLH FLH total kidney enrichment with beads CD CD5 5. FLH CD5 after FACSsort Depletion of CCR Gr with MC Depletion of CCR with MC Peripheral blood UUO Kidney Day Day Day 7, Day 7 n = MC n = 5, day : UUO day 7 : analysis, 5, day to : treatment with MC(antiCCR) or mab MC Gr Gr MC MC Gr Gr Gr monos monos No reduction of fibrocytes after treatment with MC Absence of CCR in CCRKO mice Fibrocytes in the kidney CCRKO mice MC,, n = MC n = Blood Day Day 7 WT 5,, Kidney WT n = 5 CCR / n = 5, 5, # CDb CDb Gating on CD5 cells UUO contra No reduction in Col I mrna expression Gr CCR / 5 Gr Gr monos monos WT n = 5 CCR / n = 5 5 # day day 7
7 acollagen I rabbit IgG Collagen I Rabbit IgG Gr per µl blood /7/ Reduction of fibrocytes in CCRKO mice WT CCR / 5,, 5, CDb, CD5, Col I (recovered per kidney) α Collagen I % Collagen I positive area Reduced fibrosis in CCRKO mice Fibrocytes in the kidney. WT.. : p <,5 UUO contra CCRKO. CDb Gating on CD5 cells CDb 5 WT CCR / n = 5 n = 5. WT CCR / 5 # day day 7 treatment with IL a from day treatment with IL a from day UUO kidney ILa CD5 IL a IL a CD5 IL UUO contra spleen co IL treatment with IL a from day Influence of T cells on fibrosis in UUO Analysis of BALB/c versus SCID mice IL UUO contra spleen BALB/c SCID CD5 BALB/c SCID CD5 BALB/c SCID UUO contra 7
8 α Collagen I α Collagen I α Collagen I Isotyp Isotyp Isotyp /7/ Influence of T cells on fibrosis in UUO Analysis of BALB/c versus SCID mice treatment with anticd and immunsuppressants BALB/c SCID µm µm 5 9 BALB/c SCID Cyclosporine A Rapamycin Control Cyclosporine A Rapamycin Control oil CyA Rapa CD5 CD5 oil CyA Rapa treatment with anticd and immunsuppressants Summary IL a 5 acd CNI Absence of CD T cells oil CyA Rapa 5 5 oil CyA Rapa UUO Interstitial fibrosis Absence of Gr Niedermeier et al. PNAS 9 Acknowledgment University Hospital Regensburg Marianne Niedermeier Barbara Reich Christian Weingart Katrin Schmidbauer Andrea Denzel ManuelRodriguez Gomez Nicole Goebel Yvonne Talke Matthias Mack Institut for Physiologie Frank Schweda Richard Warth
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