JOURNAL CLUB Barbara Labban, M.D.

Complement System Crucial component of the innate immune system. Crucial component of the innate immune system. - Host defense - Clearance of immune complexes and dying cells - Adjuvant role in immune response

Classical Pathway Lectin Pathway Alternative Pathway Immune Complexes Non- Immune Complexes C1 C4 C4b C2 C2a C4b2a (C3 Convertase) C1-INH C4bp fI (-) C3 C3b C4b2a3b (C5 Convertase) C5C5b +C6 +C7 +C8 +C9 C5b-9 (MAC) Endotoxin C3 C3b Factor B Properdin f D C3bBb (C3 Convertase) C3b C3bBb3b (C5 Convertase) C3

Classical Pathway Activation Ab-dependent Ag-IgM, Ag-IgG complexes Ab-Independent polyanions, protamine, DNA, RNA of apoptotic cells, G- Bacteria, CRP C1 + + C4 + C2 + C3 C 4b2a3b C1-INH ( - )

Classical Pathway Immune Complexes Non- Immune Complexes C1 C4 C4b C2 C2a C4b2a (C3 Convertase) C1-INH C4bp fI (-) C3 C3b C4b2a3b (C5 Convertase) C5C5b +C6 +C7 +C8 +C9 C5b-9 (MAC)

Lectin Pathway Ab-Independent Ab-Independent C4 Activation Microbial Carbohydrates (+) MBL +(MASP-1, MASP-2)

Alternative Pathway Continuous state of low-level activation Continuous state of low-level activation Continuous generation of C3b in plasma Continuous generation of C3b in plasma Active regulation is achieved by 2 plasma proteins: Factor H & Factor I. Active regulation is achieved by 2 plasma proteins: Factor H & Factor I.

Alternate Pathway C3 C3b C3a LPS +H2OC3(H2O) + f B C3(H2O)B f D +f B C3bB f D IgG-C3bBb-Properdin (C3 Convertase) C3(H2O)Bb (C3 Convertase) IgG-C3bB- Properdin n C3 n C3b IgG-C3b- Properdin +f B i C3b + C3f C3dg + C3c H Factor I H MCP CD46 CR1 C3NeF Stabilized, C3 Convertase. Auto-antibody attaches to convertase H H Amplification Loop H

Factor H Single chain glycoprotein. Produced by the liver. Single chain glycoprotein. Produced by the liver. Composed of 20 Short Consensus Repeat (SCR) domains. Composed of 20 Short Consensus Repeat (SCR) domains. Each SCR composed of 60 amino-acids. Each SCR composed of 60 amino-acids. N-terminal SCR domains: Regulation of fluid- phase C3 activation. N-terminal SCR domains: Regulation of fluid- phase C3 activation. C-terminal domains: Regulation of surface bound C3b (surface recognition function, depends on the chemical composition of the surface). C-terminal domains: Regulation of surface bound C3b (surface recognition function, depends on the chemical composition of the surface).

Factor H - Regulates the alternative Pathway : Inhibits the formation of the C3 Convertases of the alternative pathway by binding to C3b Inhibits the formation of the C3 Convertases of the alternative pathway by binding to C3b Promotes dissociation of the C3 Convertases (Decay acceleration activity) Promotes dissociation of the C3 Convertases (Decay acceleration activity) Cofactor to Factor I- mediated proteolytic conversion of C3b to inactive C3b (i C3b). Cofactor to Factor I- mediated proteolytic conversion of C3b to inactive C3b (i C3b). - Deficiency of Factor H  Uncontrolled alternative pathway activation with secondary depletion of C3, Factor B and properdin.

Factor H and Renal Disease Factor H gene mutation is associated with MPGN type II and atypical HUS. Factor H gene mutation is associated with MPGN type II and atypical HUS. Mutations located in the C terminal  aHUS. Rarely result in low complement or low Factor H plasma levels. Mutations located in the C terminal  aHUS. Rarely result in low complement or low Factor H plasma levels. Mutations located in the N- terminal (complement regulatory domains) or complete factor H deficiency  MPGN 2. Mutations located in the N- terminal (complement regulatory domains) or complete factor H deficiency  MPGN 2. - Caprioli et al. The molecular basis of familial hemolytic uremic syndrome: mutation analysis of factor H gene reveals a hot spot in short consensus repeat 20. J Am Soc Nephrol 2001; 12: Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50)

MPGN type II (Dense Deposit Disease) MPGN type II (Dense Deposit Disease) MPGN II: intramembranous electron-dense material along the GBM MPGN II: intramembranous electron-dense material along the GBM Positive staining for C3, C5 & C9. Positive staining for C3, C5 & C9. Cleavage fragments of C3b such as C3c and C3dg are found in the plasma of patients with MPGN2. Cleavage fragments of C3b such as C3c and C3dg are found in the plasma of patients with MPGN2. Complete Factor H deficiency in humans, pigs & mice results in uncontrolled Alternative pathway activation leading to MPGN2. Complete Factor H deficiency in humans, pigs & mice results in uncontrolled Alternative pathway activation leading to MPGN2. Appel et al. Membranoproliferative Glomerulonephritis Type II (Dense Deposit Disease) An update. J Am Soc Nephrol 16: , doi:

MPGN type II (Dense Deposit Disease) Case Report: Patient with Autoantibody to factor H  MPGN2 Case Report: Patient with Autoantibody to factor H  MPGN2 Meri S. et al. Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis. J. Exp. Med. 1992;175:939–950) Meri S. et al. Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis. J. Exp. Med. 1992;175:939–950) Case Report: Mutation of factor H complement regulatory domains  MPGN2 Case Report: Mutation of factor H complement regulatory domains  MPGN2 Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50 ) Licht C., et al. Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novel pathomechanism for dense deposit disease (MPGN II). Kidney Int. 2006;70:42–50 )

Hogasen et al. Hereditary porcine membranoproliferative glomeulonephritis type II is caused by Factor H deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, Hogasen et al studied 13 piglets affected by MPGN type II to investigate the cause of complement activation. Hogasen et al studied 13 piglets affected by MPGN type II to investigate the cause of complement activation. Plasma transfusion resulted in Plasma transfusion resulted in - increase in C3 levels - increase in median and maximum survival ( 82 and 375 d vs 37 and 72 d) - MPGN was less proliferative compared to untreated pigs Conclusion: The deficient regulatory protein was present in plasma. Conclusion: The deficient regulatory protein was present in plasma.

Hogasen et al. Hereditary porcine membranoproliferative glomerulonephritis type II is caused by Factor H deficiency. The American Society for Clinical Investigation.Vol 95, March 1995, Fractionation of normal plasma, and administration of single proteins with monitoring of the C inhibitory activity in vivo Fractionation of normal plasma, and administration of single proteins with monitoring of the C inhibitory activity in vivo Conclusion: Inhibitory activity is confined to one single protein. Conclusion: Inhibitory activity is confined to one single protein kDa kDa - Cofactor to factor I in vitro - Cofactor to factor I in vitro - NH2-terminal amino acid sequence was homologous with human, murine and rabbit FH - NH2-terminal amino acid sequence was homologous with human, murine and rabbit FH

Pickering et al. Pickering et al. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H. Nature genetics volume 31 august 2002 Pickering et al produced Factor H deficient mice and compared cohorts of Cfh-/-, Cfh+/- and wild-type mice for 8 months Pickering et al produced Factor H deficient mice and compared cohorts of Cfh-/-, Cfh+/- and wild-type mice for 8 months At 8 months: At 8 months: - 23 % mortality in Cfh -/- mice - Cfh -/- mice developed light microscopic features of MPGN II

Light microscopy: the glomerulus of Cfh–/– mice shows hypercellularity, mesangial expansion and thickening of the capillary walls with double contours.

Immunoflurescence microscopy capillary wall and mesangial deposition of C3 and C9 occurs in Cfh–/– mice. No deposition of C3 or C9 is seen in wild-type mice.

Glomerular C3 deposition preceded the appearance of GBM dense deposit which preceded the histological light microscopic appearance of MPGN. Glomerular C3 deposition preceded the appearance of GBM dense deposit which preceded the histological light microscopic appearance of MPGN. MPGN II was totally dependent on C 3 activation. MPGN II was totally dependent on C 3 activation. Generation of mice deficient in factors H and factor B prevented C3 activation and prevented the development of MPGN Generation of mice deficient in factors H and factor B prevented C3 activation and prevented the development of MPGN

Capillary wall deposition of C3 can be seen in Cfh–/–Bf+/– mice but is absent in Cfh–/–Bf–/– littermates. Age-matched healthy Cfh–/– mice show capillary wall deposition of C3 and C9. Before the development of MPGN, no IgG deposition is evident in the kidneys of Cfh–/– mice.

Pickering et al. Prevention of C5 activation ameliorates spontaneous and experimental glomerulonephritis in factor H-deficient mice. Proc Natl Acad Sci U S A June 20; 103(25): 9649–9654. In another study, Pickering et al. prevented C5 activation by crossing factor H- deficient mice with C 5 deficient animals. In another study, Pickering et al. prevented C5 activation by crossing factor H- deficient mice with C 5 deficient animals. No prevention in the development of the GBM deposits. No prevention in the development of the GBM deposits. Reduction in glomerular cellularity, serum creatinine levels and mortality. Reduction in glomerular cellularity, serum creatinine levels and mortality.

Reduction in glomerular cellularity, serum creatinine levels and mortality in Cfh-/- C5-/- mice.

Uncontrolled activation of C3 in plasma is essential for the development of MPGN II Factor H deficiency Factor H deficiency IgG autosomal Ab (): binds to and stabilizes the Alternative Pathway C3 Convertase  enhanced Alternative Pathway activation. IgG autosomal Ab (C3NeF): binds to and stabilizes the Alternative Pathway C3 Convertase  enhanced Alternative Pathway activation. Inherited dysfunctional C3 molecules: C3 convertase are resistant to inhibiton by factor H  Enhanced AP activation. Inherited dysfunctional C3 molecules: C3 convertase are resistant to inhibiton by factor H  Enhanced AP activation. - Linshaw M.A., et al. Hypocomplementemic glomerulonephritis in an infant and mother. Evidence for an abnormal form of C3. Am. J. Nephrol. 1987;7:470–477 - Schena FP et al. Biological significance of the C3 Nephritic factor in membranoproliferative Glomerulonephritis. Clin Nephrol 1982; 18:240-6

Alternative Pathway C3 C3b C3a LPS +H2OC3(H2O) + f B C3(H2O)B f D +f B C3bB f D IgG-C3bBb-Properdin (C3 Convertase) C3(H2O)Bb (C3 Convertase) IgG-C3bB- Properdin n C3 n C3b IgG-C3b- Properdin +f B i C3b + C3f C3dg + C3c H Factor I H MCP CD46 CR1 C3NeF Stabilized, C3 Convertase. Auto-antibody attaches to convertase H H Amplification Loop H

Factor I 88 k-Da serine protease 88 k-Da serine protease Function: inactivation of C3b Function: inactivation of C3b Inactivation of C3b in 2 steps: Inactivation of C3b in 2 steps: - Step 1: Factor I cleaves the α-chain of C3b at 2 sites, releasing C3f and forming i C3b. Cofactors: Factor H, MCP, CD 46, CR1. Cofactors: Factor H, MCP, CD 46, CR1. - Step 2: Factor I further cleaves iC3b to produce C3dg & C3c.

Appel et al. Membranoproliferative Glomerulonephritis Type II (Dense Deposit Disease) An update. J Am Soc Nephrol 16: , doi:

Factor I inhibits C3b  inhibits the C3 convertase of the alternative pathway  limits the alternative pathway amplification. Factor I inhibits C3b  inhibits the C3 convertase of the alternative pathway  limits the alternative pathway amplification. Factor I deficiency: Uncontrolled AP activation Factor I deficiency: Uncontrolled AP activation  Depletion of C3, Factor B & Properdin  High C3b  Reduction of Factor H levels (due to formation of factor H- C3b complexes) NEVER ASSOCIATED WITH MPGN 2 !

Study Purpose - MPGN2 has never been reported in individuals with Factor I deficiency To determine why uncontrolled C3 activation in the context of factor I deficiency does not result in MPGN II. To determine why uncontrolled C3 activation in the context of factor I deficiency does not result in MPGN II.

Cf i -/- mice were produced (disrupted the gene encoding factor I, by deleting exon 4 of the Cfi gene with a gene-targeting replacement vector). Cf i -/- mice were produced (disrupted the gene encoding factor I, by deleting exon 4 of the Cfi gene with a gene-targeting replacement vector).

Results Complement analysis C3 level C3 level Cfi -/- : Low Cfi +/ - : normal Cfi +/+ : normal Factor H level Factor H level Cfi -/- : Low Cfi +/ - : normal Cfi +/+ : normal Factor B level Cfi -/- : Low Cfi +/ - : normal Cfi +/+ : normal

Complement analysis in Cfi-/- mice Complement analysis in Cfi-/- mice Plasma C3, factor H and factor B levels in Cfi deficient, Cfi+/–, and wild-type mice. Horizontal bars denote median values. Plasma C3, factor H and factor B levels in Cfi deficient, Cfi+/–, and wild-type mice. Horizontal bars denote median values.

Western blot to determine the nature of plasma C3: C3 α chain 115 kDa C3 α chain 115 kDa C3b α’ chain 110 kDa C3b α’ chain 110 kDa ß chain kDa ß chain kDa Cfi -/- : HMW bands ( aggregates of C3b) present Cfi -/- : HMW bands ( aggregates of C3b) present C3c not detectable C3c not detectable Cfi +/+: HMW bands ( aggregates of C3b) absent Cfi +/+: HMW bands ( aggregates of C3b) absent C3c Detectable C3c Detectable

Western blot from wild-type and Cfi–/– mice: (D) α′-chain(C3b) derived from Cfi–/– plasma ran at approximately 110 kDa. α-chain(C3) derived from wild-type plasma 115 kDa (E) C3c was detectable only in plasma derived from wild- type mice, as evident by the presence of 130-kDa bands.

MPGN2 development Monitored cohorts of wild type, Cfi+/- and Monitored cohorts of wild type, Cfi+/- and Cfi-/- mice over an 8 month period at which time all were sacrificied and renal function and histology assessed. Cfi-/- mice over an 8 month period at which time all were sacrificied and renal function and histology assessed. No difference in plasma urea levels or albuminuria No difference in plasma urea levels or albuminuria No difference in glomerular hypercellularity. No capillary wall double contour. No difference in glomerular hypercellularity. No capillary wall double contour. More mesangial expansion and deposition of nodular hyaline material. More mesangial expansion and deposition of nodular hyaline material.

Light microscopic appearances of mesangial hyalinosis in 8-month-old Cfi–/– mice. PAS-stained glomerular sections from 8-month-old wild-type (A) and Cfi–/– mice (B and C). The glomerulus from the wild-type mouse had normal appearances. In contrast, areas of mesangial expansion with a nodular hyaline appearance were evident in the Cfi–/– mice (arrows). Original magnification, ×40 (B); ×100(A and C).

Glomerular C3 staining in Cfi-/- mice : Increased glomerular C3 staining in Cfi-/- mice but MESANGIAL in distribution. Increased glomerular C3 staining in Cfi-/- mice but MESANGIAL in distribution. Linear capillary wall staining pattern in age matched Cfh-/- animals. Linear capillary wall staining pattern in age matched Cfh-/- animals. Glomerular Ig G staining did not differ between Cfi-/- and wild type mice. Glomerular Ig G staining did not differ between Cfi-/- and wild type mice.

Glomerular C3 staining in 8-month-old Cfi–/– mice. (A) Increased glomerular C3 staining in Cfi–/– mice, with mesangial in distribution. (B) linear capillary wall staining pattern in Cfh–/– mice. (C) Glomerular C3 staining in Cfi+/– mice is identical to the wild-type animals (D). (E) Quantification of the glomerular C3 staining confirmed significantly increased glomerular C3 in the Cfi deficient mice

Cfi-/- mice developed increased mesangial C3 staining but not deposition of C3 along GBM Cfi-/- mice developed increased mesangial C3 staining but not deposition of C3 along GBM Factor I is an absolute requirement for GBM C3 deposition and the development of MPGN II in Cfh-/- mice Factor I is an absolute requirement for GBM C3 deposition and the development of MPGN II in Cfh-/- mice Glomerular staining patterns in Cfi-/- mice with either heterozygous or homozygous factor H deficiency Glomerular staining patterns in Cfi-/- mice with either heterozygous or homozygous factor H deficiency

Glomerular C3 staining in Cfi–/– mice with normal (Cfh+/+), heterozygous (Cfh+/–), and homozygous (Cfh–/–) factor H genotypes. A mesangial staining pattern was evident in the glomeruli of the Cfi–/– mice regardless of factor H genotype.

Factor I deficiency prevented accumulation of C3 along the GBM in Cfh-/- mice. Factor I deficiency prevented accumulation of C3 along the GBM in Cfh-/- mice. Light Microscopy: No evidence of MPGN2 Light Microscopy: No evidence of MPGN2 in 8 month old Cfh-/- Cfi-/- mice. in 8 month old Cfh-/- Cfi-/- mice.

Factor H deficient mice (cfh -/-) Factor H deficient mice (cfh -/-) - Florid C3 deposition along GBM/ MPGN2 occurred Factor I deficient mice (cfi -/-) Factor I deficient mice (cfi -/-) Uncontrolled AP Uncontrolled AP - Did not develop C3 deposition /MPGN2 Factor H & Factor I deficient (cfh -/-, cfi -/-) Factor H & Factor I deficient (cfh -/-, cfi -/-) Uncontrolled AP Uncontrolled AP - Did not develop C3 deposition/MPGN2

Plasma C3 (B) and factor B (C) levels in Cfi–/– mice with heterozygous and homozygous factor H deficiency. C3 level is lowest in Cfh-/- mice. Factor B level is equally low in all types. Plasma C3 (B) and factor B (C) levels in Cfi–/– mice with heterozygous and homozygous factor H deficiency. C3 level is lowest in Cfh-/- mice. Factor B level is equally low in all types.

Plasma analysis: Plasma analysis: (cfi -/-): C3b (cfh -/-) : C3b cleavage products (cfi -/-, cfh -/-): C3b Addition of Factor I: Addition of Factor I: (cfi-/-),(cfi-/-,cfh-/-) + Factor I  C3b fragments in plasma. Plasma analysis Plasma analysis

Western blot for C3 from Cfi–/– and Cfi–/–Cfh–/– mice under reducing conditions show α′- chain and HMW bands. Fragments were only present in the Cfh–/– EDTA plasma. Sera from Cfi–/– and Cfi–/–Cfh–/– mice before and after incubation with murine sera deficient in C3 (as a source of autologous factors I and H) show complete cleavage of the α′- chain with the concomitant appearance of the α′-chain fragment

Under nonreducing conditions C3c was detectable in EDTA plasma from Cfh–/– and wild-type mice, but not from Cfi–/– mice.

Following injections of sera from Cfh–/–C3–/– mice (used as a source of autologous factor I) to mice with combined deficiency of factor H and factor I. plasma C3 levels fell rapidly to levels comparable with those seen in mice with factor H deficiency alone

Infusion of serum containing factor I ( sera from Cfh-/-C3-/- mice) to mice with combined deficiency of factor H and factor I. Appearance of α chain fragments in the circulation.

Florid glomerular C3 staining identical to that seen in unmanipulated Cfh–/– mice was present in the reconstituted animals, in marked contrast to the mesangial C3 staining present in the noninjected Cfh–/–Cfi–/– control mice.

The Cfh–/– mice with heterozygous factor I deficiency had identical glomerular C3 staining to that seen in Cfh–/– animals. Also had markedly reduced C3 levels (median 17.8) comparable with mice deficient in Cfh-/- alone.

Factor I is critically required for GBM C3 deposition in Cfh-/- mice. Factor I is critically required for GBM C3 deposition in Cfh-/- mice. The C3 deposited along the GBM in factor H deficient mice derives from the circulation: The C3 deposited along the GBM in factor H deficient mice derives from the circulation: Examined glomerular staining 6 weeks post kidney transplant: - wild-type mice into Cfh-/- recipients - wild-type mice into Cfh-/- recipients - Cfh-/-,C3-/- mice into Cfh-/- recipients - Cfh-/- mice into Cfh-/- recipients - wild-type mice into wild-type recipients

(A), (B) & (D): Linear capillary wall C3 staining. kidneys transplanted into Cfh–/– animals. (C): No abnormal glomerular C3 staining. (A), (B) & (D): Linear capillary wall C3 staining. kidneys transplanted into Cfh–/– animals. (C): No abnormal glomerular C3 staining.

Conclusion In Cfi–/– mice, factor H is not responsible for the lack of GBM C3 deposition. In Cfi–/– mice, factor H is not responsible for the lack of GBM C3 deposition. Factor I is a critical requirement for GBM C3 deposition and subsequent MPGN2 in Cfh–/– mice. Factor I is a critical requirement for GBM C3 deposition and subsequent MPGN2 in Cfh–/– mice. During uncontrolled alternative pathway activation, it is the nature of the plasma C3 activation product that determines whether GBM C3 deposition develops. During uncontrolled alternative pathway activation, it is the nature of the plasma C3 activation product that determines whether GBM C3 deposition develops.

Conclusion The important difference between factor I and factor H deficiency is the fate of plasma C3b: The important difference between factor I and factor H deficiency is the fate of plasma C3b: - Factor I deficiency: No Cleavage products No MPGN No MPGN - Factor H deficiency: Cleavage products present MPGN develops MPGN develops Factor I–mediated cleavage of C3b to form iC3b, C3c, and C3dg in Factor H deficiency occurs as a consequence of alternative factor I cofactors in the circulation (CR1 on erythrocytes, plasma factor H–like protein 1 and plasma factor H–related protein 5). Factor I–mediated cleavage of C3b to form iC3b, C3c, and C3dg in Factor H deficiency occurs as a consequence of alternative factor I cofactors in the circulation (CR1 on erythrocytes, plasma factor H–like protein 1 and plasma factor H–related protein 5).

Conclusion There remains no definitive therapy for MPGN2. There remains no definitive therapy for MPGN2. Prevention of C5 activation ameliorated but did not prevent the development of MPGN2 in Cfh–/– mice. Prevention of C5 activation ameliorated but did not prevent the development of MPGN2 in Cfh–/– mice. GBM C3 deposition secondary to alternative pathway dysregulation could be prevented, regardless of etiology, by strategies that either prevent proteolytic cleavage of C3b or sequester its metabolites in the circulation. GBM C3 deposition secondary to alternative pathway dysregulation could be prevented, regardless of etiology, by strategies that either prevent proteolytic cleavage of C3b or sequester its metabolites in the circulation.