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MANNOSE-BINDING LECTIN PATHWAY

MANNOSE-BINDING LECTIN PATHWAY. Gary C. Pien, MD/PhD Division of Allergy/Immunology Children’s Hospital of Philadelphia 34 th St and Civic Center Blvd Philadelphia, PA 19104. COMPLEMENT ACTIVATION PATHWAYS. Janeway, C, et al, Immunobiology , New York: Garland Science, 2005.

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MANNOSE-BINDING LECTIN PATHWAY

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  1. MANNOSE-BINDING LECTIN PATHWAY Gary C. Pien, MD/PhD Division of Allergy/Immunology Children’s Hospital of Philadelphia 34th St and Civic Center Blvd Philadelphia, PA 19104

  2. COMPLEMENT ACTIVATION PATHWAYS Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  3. MANNOSE-BINDING LECTIN (MBL) • pattern recognition receptor • recognizes pathogen-associated molecular patterns (PAMPs) • member of collectin sub-family of C-type lectins, such as surfactants A/D • serum protein • synthesized primarily in liver • “acute phase reactant” • rises up to 3x in 1-2 weeks • multimers of homotrimers • collagen-like helix domain (stalk) • -helical coiled coil (neck) • carbohydrate-recognition domain (CRD) • structurally homologous to C1q Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  4. MBL STRUCTURE AND FUNCTION • binding sites recognize D-mannose, L-fucose, N-acetylglucosamine • binding sites have fixed orientation and spacing (45 Å) • ligands must have correct orientation and spacing • provides “antigen specificity” • structures “found only on microbes and not on host cells” – overstated? Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  5. MBL LIGANDS Worthley, DL, et al, 2006, World J Gastroenterol 12:6420; Takashi, K, et al, 2006, Curr Opin Immunol 18:16.

  6. MBL OPSONIZATION • MBL pathway homologous to classical complement pathway • acts as an “ante-antibody” • MBL binds target carbohydrates on microbial surfaces • recruits MBL-associated serine proteases (MASPs) to site • MBL:MASP complex activates complement, leading to downstream anti-microbial functions Takashi, K, et al, 2006, Curr Opin Immunol 18:16.

  7. MASPs – 2 GENES, 4 PRODUCTS • MASP1 • can cleave C2, somewhat C3 • function unknown • MASP3 • alternative splice product of MASP1 gene • lacks serine protease domain • function unknown • MASP2 • minimal functional unit = MASP2 homodimer + two MBL homotrimers • cleaves C4 and C2 to generate C3 convertase (C4bC2b) • MAp19 • alternative splice product of MASP2 gene • function unknown Sorensen R, et al, 2005, Springer Semin Immunopathol 27:299.

  8. MBL:MASP “TULIPS” • MASPs are predominantly synthesized in liver • form Ca+2-dependent homodimers • unknown why heterodimers do not occur • MASP1 and MAp19 associate with low oligomers of MBL • MASP3 and MASP2 associate with higher order oligomers • MASPs can also associate with ficolins (another group of PAMP binders) • binding of MBL:MASP complex to ligand activates serine protease • unclear mechanism for how this occurs Sorensen R, et al, 2005, Springer Semin Immunopathol 27:299; Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  9. COMPLEMENT ACTIVATION • activated MBL:MASP cleaves C4, C2 to form C3 convertase • C3 convertase is bound to surface of pathogen • cleaves other factors to produce effector functions Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  10. MEMBRANE-ATTACK COMPLEX C5b, C6, C7, C8, C9 Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  11. AGE AND MBL LEVELS • at birth, MBL level is 2/3 of adult concentration • adult level reached at 1 month of age • small decline in levels through adulthood Ip WK, et al, 2004, Scand J Immunol 59:310.

  12. GENETIC VARIATION • MBL encoded by MBL2 gene • MBL1 pseudogene • both closely positioned on chromosome 10 • exons 1-2 encode Gly-Xaa-Yaa triple collagen helix motif to form stalk • exon 3 encodes coiled-coil neck • exon 4 encodes CRD domain • polymorphisms exist in exon 1 that affect MBL serum levels • promoter polymorphisms also exist that affect expression levels Garred, P, et al, 2006, Genes and Immunity 7:85.

  13. GENETIC VARIATION • A is normal allele  A/A is normal haplotype • B, C, and D are variant alleles • O indicates any of the variant alleles • serum MBL levels correlate with haplotype • however, people with same genotype may differ in MBL levels by 10x Garred, P, et al, 2006, Genes and Immunity 7:85; Worthley, DL, et al, 2006, World J Gastroenterol 12:6420

  14. GENETIC VARIATION • promoter polymorphisms at 3 distinct locations: H/L, X/Y, P/Q • 7 possible MBL haplotypes  28 possible genotypes (diploid) • HYPA • LYPA • LYQA • LXPA • HYPD • LYPB • LYQC Garred, P, et al, 2006, Genes and Immunity 7:85; Thiel, S, et al, 2006, Mol Immunol 43:86;Kilpatrick, DC, Biochimica 2002, 1572:401.

  15. GENETIC VARIATION • promoter polymorphisms at 3 distinct locations: H/L, X/Y, P/Q • 7 possible MBL haplotypes  28 possible genotypes (diploid) Dommett, RM, et al, 2006, Tissue Antigens 68:193.

  16. GENETIC VARIATION • variant MBL alleles have alterations in collagen helix stalk • impaired multimerization • forms low-order oligomers • oligomers have preference for MASP1 and MAp19 … cascade failure? • non-functional and unstable Eisen, DP and Minchinton RM, 2003, Clin Inf Dis 37:1496.

  17. MBL LEVELS CORRELATE WITH ACTIVITY • MBL concentration affects activity of complex and C4b deposition Worthley, DL, et al, 2006, World J Gastroenterol 12:6420

  18. MBL DEFICIENCY AND DISEASE ASSOCIATIONS • MBL linked to human disease based on case report in 1968 • female patient with recurrent URI and diarrhea in childhood • no apparent immunodeficiency • neutrophils cultured in patient’s serum could not phagocytose yeast • low C3 deposition but normal complement function • “common opsonic defect” with impaired complement deposition • defect found in 5-8% of health population • separately, group purified a protein from rabbit liver using yeast mannan • protein could activate complement when bound to mannan surfaces • led to identification of MBL Garred, P, et al, 2006, Genes and Immunity 7:85.

  19. MBL DEFICIENCY AND DISEASE ASSOCIATIONS • what is a physiologically “low” level? • ranges from 5 – 10,000 ng/ml in population • no individual completely deficient in MBL has been reported • 30% of population has level < 500 ng/ml • 10-20% have levels < 100 ng/ml • 50-100 ng/ml has been labeled “severe MBL deficiency” • majority of individuals with low MBL are healthy • difficult to link MBL deficiency with infection susceptibility • infants have recurrent respiratory infections, OM, chronic diarrhea • MBL important when maternally acquired antibodies wane and host responses still maturing? • clinical significance likely varies depending on disease context • disease susceptibility modifier? Garred, P, et al, 2006, Genes and Immunity 7:85.

  20. MBL DEFICIENCY AND INFECTIONS Eisen, DP and Minchinton RM, 2003, Clin Inf Dis 37:1496.

  21. MBL DEFICIENCY AND INFECTIONS Eisen, DP and Minchinton RM, 2003, Clin Inf Dis 37:1496.

  22. MBL DEFICIENCY AND INFECTIONS Eisen, DP and Minchinton RM, 2003, Clin Inf Dis 37:1496.

  23. MBL DEFICIENCY AND DISEASE ASSOCIATIONS • chemotherapy patients • children with ALL and variant MBL had twice as many days of F&N • adults who developed bacteremia or pneumonia after chemotherapy had lower levels of MBL than those who did not • low MBL associated with increased rates of severe infections and F&N in adult cancer patients • allogeneic hematopoietic stem cell transplant patients • invasive infections occurred more frequently in patients with variant MBL alleles Thiel, S, et al, 2006, Mol Immunol 43:86.

  24. MBL DEFICIENCY AND DISEASE ASSOCIATIONS • SIRS / sepsis / MOD • adults who died in ICU from sepsis/MOD had 3x lower MBL levels • adults with variant MBL genotypes had higher risk of sepsis Thiel, S, et al, 2006, Mol Immunol 43:86; Klein, NJ, 2005, Mol Immunol 42:919.

  25. MBL DEFICIENCY AND DISEASE ASSOCIATIONS • autoimmunity • low MBL levels linked to worse prognosis in RA • low MBL predisposes to SLE • low MBL strongly associated with more severe celiac disease • vascular disease • higher frequency of MBL mutations in patients with Kawasaki disease • higher risk of MI in patients with low MBL levels • obstetrics • couples with recurrent spontaneous abortion had higher frequency of MBL deficiency in both partners • other studies only found association with maternal deficiency Thiel, S, et al, 2006, Mol Immunol 43:86.

  26. MASP DEFICIENCY • patient with MASP2 deficiency • healthy until age 13 • developed UC • erythema multiforme bullosum • SLE? • 3 episodes of severe pneumococcal pneumonia • progressive lung fibrosis • hypocomplementemia • homozygous D105G mutation in MASP2 • other family members healthy • had low activity levels in MBL pathway • were heterozygous for MASP2 mutation Stengaard-Pedersen, K, et al, 2003, NEJM 349:554.

  27. MASP DEFICIENCY • 10% of general (Danish) population is heterozygous for D105G mutation (492 pts) • 50% of normal circulating MASP2 levels (heterozygotes ~ 200 ng/ml) • individuals who are homozygous expected at 1:1000 • have only 10% of normal circulating MASP2 levels (~ 50-75 ng/ml) • no Hong Kong Chinese carried D105G mutation (573 pts) • did find another deficiency-associated mutation, rare frequency Kilpatrick, DC, Biochimica et Biophysica Acta 2002, 1572:401; Sorensen R, et al, 2005, Springer Semin Immunopathol 27:299.

  28. COMPLEMENT DEFICIENCIES - KATE http://www.clinimmsoc.org/teaching/tips/complement_deficiencies.pdf

  29. PATHWAY DEFECTS AND HUMAN DISEASE Janeway, C, et al, Immunobiology, New York: Garland Science, 2005.

  30. MBL PATHWAY TESTING • IBT Laboratories (www.ibtreflab.com) • MBL protein • sandwich ELISA • uses monoclonal antibody against oligomeric CRD • define normal > 100 ng/ml • MBL pathway function test • ELISA C4b deposition assay • functional assay of initial pathway (MBL, MASP) • ProGenotyper MBL panel • rtPCR for MBL variant alleles • ProGenotyper MASP2 polymorphisms • DNA sequencing (D105G or entire gene?)

  31. MBL REPLACEMENT THERAPY • NatImmune A/S (www.natimmune.com) • Danish biotech company • developed rhMBL • Phase I clinical trial in 2004 • infused rhMBL into healthy subjects • no significant adverse events • serum t½ = 30 hours • Enzon Pharmaceuticals (www.enzon.com) • US biotech company licensed rhMBL from NatImmune in 2005 • Phase I/II clinical trials • rhMBL prophylaxis and treatment • oncology and liver transplant patients • reduce risk of infection

  32. CONCLUSIONS • MBL pathway involved in host defense and possibly other functions • MBL deficiency alone not clearly associated with infection risk • deficiency most clearly associated with increased infection risk in immunosuppressed/co-existing immunodeficient patients • deficiency may predispose to infections in infancy/early childhood • low MBL may be susceptibility risk factor for other diseases • MASP2 deficiency has been associated with recurrent infections • n = 1 (maybe 2) • MASP2 may have other functions outside of MBL pathway (ficolins) • MASP2 deficiency may have more prominent phenotype than MBL deficiency

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