ANCA endothelial cells and neutrophils

Size: px

Start display at page:

Download "ANCA endothelial cells and neutrophils"

Tiffany Wells
5 years ago
Views:

1 ANCA endothelial cells and neutrophils Philippe Lesavre Université Paris Descartes INSERM U 845 Hôpital Necker, Paris

2 small renal vessels glomeruli small vessels interlobular 50 µm medium size arcuate 70 µm interlobar 100 µm cliché L Bankir et JP Grünfeld

3 glomerular and peritubular capillaries 1. glomerular capsule 2. glomerulus 3. afferent arteriole 4. efferent arteriole 5. proximal convoluted tubule 6. distal convoluted tubule 7 = collecting duct 8 = loop of Henle 9 = peritubular capillaries (or vasa recta)

4 ANCA-associated glomerular capillaritis focal necrosis Noël LH

5 glomerular and peritubular capillaries 1. glomerular capsule 2. glomerulus 3. afferent arteriole 4. efferent arteriole 5. proximal convoluted tubule 6. distal convoluted tubule 7 = collecting duct 8 = loop of Henle 9 = peritubular capillaries (or vasa recta)

6 ANCA-associated peritubular capillaritis tubulo-interstitial nephritis - associated with ANCA vasculitis - overt in GPA - cause of reversible ARF - in absence of glomerular lesions in some cases Noël LH Wen Y K et al., Ren Fail, 2006, 28; 189 Nakabayashi K et al., Clin Exp Nephrol,2009, 13

7 the four ANCA vasculitis NCGN micropa Wegener + CSS kidney : hematuria, proteinuria (1 g/l) rapidly progressive renal failure + lung, skin, eyes neuritis/cns arthritis, muscles + granulomas (lung, ENT) + atopy, late asthma eosinophilia

8 neutrophil granules SECRETORY VESICLES AZUROPHILIC GRANULES Proteinase 3 Elastase Cathepsin G Azurocidin Myeloperoxidase BPI Defensins CR1 CR3 Albumin Proteinase 3 SPECIFIC GRANULES Collagenase Gelatinase Lactoferrin CR3 Proteinase 3

9 azurophilic granule proteinase 3 MPO ethanol fixation canca proteinase 3 panca MPO

10 ANCA in systemic vasculitis specificity (1-FP) ELISA IF PR3 MPO IF + ELISA 76 % 88 % 91 % 98 % EEC/BCR 1998

11 ANCA specificities PR3 MPO NCGN kidney + micropa Wegener + CSS lung, skin, eyes neuritis/cns arthritis, muscles + granulomas (lung, ENT) + atopy, late asthma eosinophilia

12 ANCA european gradient Wegener PR3 ANCA PR3 ANCA MPO micropa MPO

13 patients (n) Count ANCA vasculitis: age distribution n = Diagnosis code 1 2 MPA Wegener's Age (Banded) age EUVAS

14 ANCA vasculitis: relapses 1.00 n = 550 MPA 33% MPA 0.50 WG 66% Wegener's follow up 10 yr EUVAS

15 ANCA negative vasculitis Eisenberger Chen patients CGN/MPA/Weg M/F age constitutional symptoms severity n=20 1/17/2 15/5 65 yr (33-82 yr) 35% lower activity higher chronicity n=28 16/ yr (15-65 yr) 21% (66%) higher severity lower extra renal Eisenberger U et al, Nephrol Dial Transplant, 2005 Chen M et al, J Am Soc Nephrol, 2007

16 neutrophil/endothelium interactions are highly regulated innate immunity neutrophil migration towards inflammation site Toxic proteases and oxidants Phagocytosis no premature release of toxic oxidants and enzymes neutrophil adhesion restricted to microvessels adjacent to inflammation site

17 move, stop, migrate and kill with the sense of time and space post capillary venules

18 ANCA-associated vasculitis unwanted premature neutrophil activation in microvessels due to dysregulation of neutrophil activation and of neutrophil/endothelial interactions

19 experimental anti-mpo-induced GN Vasculitic lesions - are due to neutrophils - are amplified by LPS - involve TNF- Huugen et al. Am J Pathol 2005

neutrophils in plasma - neutrophil priming (TNF) = hyper-responsiveness to chemoattractants or IC - in whole blood, neutrophil responses to low levels of inflammatory stimuli are prevented TNF-

20 neutrophils in plasma - neutrophil priming (TNF) = hyper-responsiveness to chemoattractants or IC - in whole blood, neutrophil responses to low levels of inflammatory stimuli are prevented TNF- Ceramide Fuortes J Leukoc Biol CD43 ANCA plasma albumin anti-oxidants proteins protease-inhibitors (AAT) - ANCA do not bind to resting neutrophils in whole blood. Abdel-Salam Kidney Int 2004 PMN + anti-pr3 ANCA Control IgG PMN + anti-pr3 ANCA + plasma mpr3

21 ANCA-associated vasculitis : role of the C Alternative Pathway No anti-mpo induced GN in Factor B- or C5-deficient mice Xiao H Am J Pathol 2007, Huugen Kidney Int 2007 TNF-stimulated neutrophils activate the C alternative pathway. Camous et al., Blood, 2011

22 complement activation by TNF-activated neutrophils TNF- Properdin C3b C3b,Bb,P C3 C5 C5a Cell-bound Properdin Resting TNF Cell-bound C3 (MFI) on TNF-activated neutrophils Classical Lectin C2 C3 Complement pathways C4 C3/C5 convertases C3 Alternative B C5 0 0 NHS AB-EDTA AB B-depl B C2-depl NHS EDTA depl C2 depl Cell-bound C3b C3 C5a Camous et al., Blood, 2011

complement activation : positive feedback amplification of neutrophil activation TNF- Properdin C3b C3b,Bb,P C3 C5 C5a C5a further activates neutrophils Classical Lectin C2

23 complement activation : positive feedback amplification of neutrophil activation TNF- Properdin C3b C3b,Bb,P C3 C5 C5a C5a further activates neutrophils Classical Lectin C2 Complement pathways C4 C3 Alternative B 150 CD11b (MFI) C3 C3/C5 convertases C Cell-bound C3b C3 C5a 0 AB-EDTA AB B-depl C2-depl NHS EDTA NHS B depl C2 depl Camous et al., Blood 2011

24 ANCA associated vasculitis role of the leucocyte 2 integrins TNF- together with C5a activates leucocyte M 2 (Mac-1) integrins which could promote transient homotypic aggregation TNF- ICAM-3 ic3b M 2 integrin C5a intracellular signaling triggered by 2-integrin engagement

ANCA associated vasculitis role of ANCA and Fc RII receptors TNF- together with 2 integrin engagement triggers signals, which enhance the membrane expression of ANCA

25 ANCA associated vasculitis role of ANCA and Fc RII receptors TNF- together with 2 integrin engagement triggers signals, which enhance the membrane expression of ANCA antigens. TNF- ICAM-3 C5a ic3b Fc RII ANCA M 2 integrin some ANCA antibodies interact with their antigen (PR3, MPO) through Fab and with neutrophil Fc receptors through Fc

Neutrophil sequestration in microvessels

26 Neutrophil sequestration in microvessels capillaries diameter: 7m / neutrophil diameter : m ANCA-induced: Actin polymerization and inceased cell rigidity Stabilization of neutrophil 2-mediated homotypic aggregation physical trapping of neutrophils

27 glomerular and peritubular capillaries 1. glomerular capsule 2. glomerulus 3. afferent arteriole 4. efferent arteriole 5. proximal convoluted tubule 6. distal convoluted tubule 7 = collecting duct 8 = loop of Henle 9 = peritubular capillaries (or vasa recta)

28 glomerular and peritubular capillaries 1. glomerular capsule 2. glomerulus 3. afferent arteriole 4. efferent arteriole 5. proximal convoluted tubule 6. distal convoluted tubule 7 = collecting duct 8 = loop of Henle 9 = peritubular capillaries (or vasa recta)

29 ANCA promote firm adhesion and transmigration ANCA Halbwachs L and Lesavre P, JASN, 2012

30 pro-adhesive role for ANCAs ANCAs promote conversion from rolling to firm arrest (flow chamber adhesion assay) Radford DJ et al., Arthritis Rheum. 2000;43:1337 Radford DJ et al., Arthritis Rheum. 2001;44:2851 Calderwood JW et al., J Leukoc Biol. 2005;77:33 anti-mpo antibodies (human) enhance chemokine-induced neutrophil adhesion and transmigration in rat mesenteric postcapillary venules Little MA et al., Blood. 2005;106: Passive transfer of antibodies against murine MPO increase cytokine-induced leukocyte adhesion in murine cremasteric postcapillary venules Nolan SL et al., J Am Soc Nephrol. 2008;19:973

31 Clustering, in lipid rafts, of neutrophil receptors required for ANCA-mediated responses. Rapid increase of ANCA-antigen membrane expression Halbwachs L and Lesavre P, JASN, 2012

32 ANCA induce the membrane expression of their own antigen during cell adhesion resting TNF no adhesion (EDTA) IgG1 TNF TNF TNF adhesion (integrins) 2 integrins TNF adhesion and ANCA TNF ANCA Fc R anti-pr3 S Brachemi al., J Am Soc Nephrol, 2007; 18: 2330

33 ANCA-bound Fc R in synergy with TNF and 2-integrin promotes massive degranulation Proteases O 2, ROI ICAM-1 TNF- C5a CP + AP C3b P C5a, C5b9 Endothelial cells

towards inflammation site Toxic proteases and

34 neutrophil/endothelium interactions are highly regulated innate immunity neutrophil migration towards inflammation site Toxic proteases and oxidants Phagocytosis recruitment of adaptive immunity and NETs formation

35 innate/adaptive immunity dendritic cell Fc- R 2 integrin cathepsin G pro-chemerin chemerin TNF- IFN- BLyS macrophages B-cell T-cell

36 B cell infiltrates in target tissues : tertiary lymphoid strutures ANCA vasculitis and lupus nephritis compartmentalized B cell clusters in the kidney with central follicular dendritic cell networks autoimmune diabetes tertiary lymphoid structures in the pancreas promote selection of B lymphocytes Steinmetz, O. M., Kidney Int; 2008 Kendall PL, J Immunol; 2007

37 intrarenal B cell cluster in ANCA vasculitis mirrors the germinal center of lymph nodes T cell B cell follicular dendritic cell T cell B cell fdc Steinmetz OM, Kidney Int; 2008

38 NETosis LL37 PR3 Fc- R MPO 2 integrin Brinkman and Zychlinsky, Beneficial suicide: when neutrophils die to make NETs, Nat Rev Microbiol, 2007 Kessenbrock et al., Netting neutrophils in auto-immune SVV Nature Med, 2009

39 netosis in ANCA vasculitis MPO-DNA in plasma Kessenbrock et al., Netting neutrophils in auto-immune SVV Nature Med, 2009

40 double positivity for MPO-ANCA and anti-gbm 5% of all ANCA-positive serum anti-gbm positive 32% of all anti-gbm positive are ANCA positive 27 patients with both antibodies, 82% had MPO-ANCA - pulmonary hemorrhage occurred in 44%. - extensive glomerular cellular crescents in most patients. - 68% of patients were dialysis-dependent at presentation - at one year (none recovered renal function) - patient survival rates 52% - renal survival 26% Levy JB et al., Kidney Int Chen M, et al., Nephrol Dial Transplant Rutgers A et al., Am J Kidney Dis. 2005

41 T cell B cell anti-gbm DC T cell B cell DC ANCA

42 asymptomatic ANCA associate with future anti-gbm disease Olson, SW, et al.,j Am Soc Nephrol, 22: , 2011.

43 T cell B cell anti-gbm DC T cell B cell DC ANCA

44 Summary Vaculitis lesions produced during ANCA-induced neutrophil activation result from the simultaneous recruitment of four simultaneous signaling: neutrophil preactivation by TNF activation of complement, releasing C5a 2 integrin engagement during homotypic aggregation and adhesion to microvascular endothelium Fc R recruitment by ANCA interacting with cell-bound antigens

45 These events result in neutrophil misleading and maximal activation within microvessels leading to endothelial lesions, recruitment of adaptive immunity and NETs formation

51 ANCA promote firm adhesion and transmigration ANCA

52 synergy of intracellular signals to neutrophils TNF- ANCA-induced Fc R clustering resting PMN TNF- Cell adhesion 2-integrin M 2 Fc -RIIa 2 integrin ICAM-1 ANCA Fc -RIIa PR3 membrane expression of ANCA antigens Degranulation Oxidative burst

53 Effects of neutrophil serine proteases (HLE, catg, PR3) on endothelial cells Cytolysis, Detachment Ballieux 1994 Enhanced apoptosis Taekema-Roelvink 1998 Enhanced IL-8 synthesis Berger 1996 Tissue factor stimulation Haubitz 2001

54 ANCA enhances endothelial damages promoted by adherent neutrophils Proteases apoptosis EC lesions EC detachment O 2, ROI ICAM-1 TNF- C5a CP + AP C3b P C5a, C5b9 Endothelial cells increased permeability

55 ANCA-associated vasculitis : role of the inflammatory cytokines Fluid phase inflammatory stimuli = early misleading signals, which give wrong information to neutrophils suggesting intravascular infection TNF- Increased levels of circulating TNF- and IL-8 (Noronha Kidney Int 1993, Ohlsson Mediators Inflamm 2004) may overcome the protective effect of plasma Anti-TNF therapy (infliximab) results in complete or partial remission in Wegener s granulomatosis or Churg-Strauss syndrome patients (Bartolucci 2002; Lamprecht 2002,Arbach 2002)

56 Effect of ANCA on adherent TNF-activated neutrophils TNF- C5a CP + AP C3b P C5a, C5b9 ICAM-1 Endothelial cells Complement activation by both classical and alternative pathway C5a and C5b-9 promote endothelial cell retraction and endothelium permeability

leucocytoclastic vasculitis

leucocytoclastic vasculitis cell infiltration (apoptotic neutrophils) necrosis of the vessel wall thrombosis cliché L-H Noël glomerular capillaritis focal necrosis cliché L-H Noël small renal vessels glomeruli