1. Methods Introduction Results Collagen XVII-specific autoantibodies in sera from patients with Parkinson's disease reacts with the neuronal, but not the epidermal, form of collagen XVII Grant Randall 1, Samuel Connell 3, Nandakumar Narayanan 4, Kelly Messingham 3, Janet Fairley 2,3 University of Missouri Kansas City School of Medicine, Medical Student 1, Kansas City, MO 64108 The Iowa City Veterans Administration 2 and Departments of Dermtology 3 and Neurology 4 at the University of Iowa Hospitals and Clinics, Iowa City, IA 52245 Conclusions Transmembrane domain NH 2 COOH NC16C sec180 NC16A 1.De Lau LM, Breteler MM. Epidemiology of Parkinson’s disease. Lancet Neurol. 5, 525–535, 2006. 2.Davie, C. (2008). A review of Parkinson's Disease. British Medical Bulletin, 86(1), 109-127. Retrieved December 6, 2015, from http://bmb.oxfordjournals.org/content/86/1/109.full 3.Fearnley JM, Lees AJ. Ageing and Parkinson’s disease: Substantia nigra regional selectivity. Brain 114, 2283–230, 1991. 4.Colbert RL, Allen DM, Fairley JA. Mortality rate of bullous pemphigoid in a U.S. medical center. J Invest Dermatol 122:1091-1095, 2004 5.Diaz, L., Ratrie, H., Saunders, W., Futamura, S., Squiquera, H., Anhalt, G., & Giudice, G. (1990). Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome. Journal of Clinical Investigation J. Clin. Invest., 1088-1094. 6.Seppänen, A. (2013). Collagen XVII: A Shared Antigen in Neurodermatological Interactions? Clinical and Developmental Immunology, 1-7. doi:10.1155/2013/240570 This work was supported in part by grants from the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development, 1I01CX000317-01 (JF), The Aging Mind and Brain Institute at the University of Iowa (NN,JF). We thank Julie McKillip, RN, and Samuel Connell BS for their assistance with the IRB and collection of patient samples. Anti-Col XVII antibodies in the PD patients were of the IgG1 subclass The anti-Col XVII antibodies from PD patients stain TH+ neurons in rat and human substantia nigra Staining of human BMZ by PD autoantibodies could not be detected by serum or by affinity concentrated IgG from PD. Nor did complement fixation assay show any BMZ specific staining. This data supports the hypothesis that the original break in tolerance to Col XVII occurs against neuronal protein in BP patients with pre-existing neurologic disease. Further it suggests that the PD epitope is cryptic in the skin., which is consistent with the lack of skin disease in these patients. BP may develop in susceptible patients due to epitope spreading to the pathogenic NC16A domain. Background Studies References & Acknowledgements B. C. D. Figure 1: Bullous pemphigoid and PD serum reactivity to Col XVII. Inflammation and blistering (A) with histologic subepidermal split (B). Schematic depicting the regions of Collagen XVII (C). BP antibodies bind the NC16A domain; the region targeted in PD is unknown. (D) Immunoblot data depicting serum reactivity to the sec180 region of Col XVII. N=23 PD sera normalized to age-matched control sera without neurologic disease. The control average is indicated by the solid line ± 95% CI. 7/25 PD sera exhibited significant (p 2 SD above negative control). Figure 2: Immunofluorescent staining reveals PD serum reactivity to dopaminergic (TH + ) neurons. PD sera (green) and murine antibodies specific for tyrosine hydroxylase (TH, red) were used to immunostain rat substantia nigra. Using a co- localization tool in ImageJ, white color was assigned to the merged image where overlap of the green and red signals was detected. Colocalization is observed using PD serum (A), but not control serum(B). Removal of sec180 specific antibodies via absorption with recombinant protein abolished the colocalization with TH (C). Mock absorption with an irrelevant protein had no effect (D). Control/TH merge PD/TH merge PD/TH Merge mock PD/TH Merge - sec180 A. B. C. D. IgG antibody purification of PD serum (right): Patient IgG was purified and concentrated using a protein G immunoaffinity column and Centricon® concentrators. The resulting purified IgG was evaluated for BMZ staining or complement fixation via indirect immunofluorescence. Sec180 protein expression and purification: The recombinant sec180 domain of collagen XVII was produced in EBNA HEK 293 cells. IgG was purified from polyclonal serum of a rabbit immunized with protein gel bands containing sec180 and the resulting antibodies were used to generate an immunoaffinity column specific for sec180 protein to allow large-scale purification of sec180 protein from supernatants. IgG subclass determination: IgG subclass was evaluated via dot-blot against sec180. A dot blot containing serial dilutions of sec180 was probed with patient sera followed by subclass specific HRP-conjugated secondary antibodies and ECL. All sera were compared to positive and negative controls. Add serum to beads to bind IgG Remove serum Add wash buffer Add elution buffer Remove wash buffer Remove elution buffer and retain Fig. 3A. Immunofluorescent staining: Indirect immuno-fluorescent staining was performed on cryosections of human foreskins obtained by routine circumcision. Sections were incubated with patient sera or purified antibodies and, after washing, reactivity was visualized using anti-IgG-488, or incubation with fresh serum followed by anti-C3-488 (green), and DAPI (blue) nuclear stain and epifluorescent microscopy. A fresh source of complement was provided in the complement testing. A positive test is shown in Fig. 3B. Results Figure 4: PD IgG does not bind to the BMZ of human skin. Cryosections of human skin were incubated with sera diluted 1:10 or purified and 5x concentrated IgG (neat). A) BP sera shows BMZ staining (positive control). B- D) Three individual sera from PD patients positive for Col XVII reactivity by immunoblot did not bind the BMZ. Likewise, purified and concentrated IgG from these same patients (F-G) did not bind the BMZ of skin. Purified IgG from a BP patient (1:10) was positive (E). A. B. C. D. E.F. G. H. B. C. D. E. Figure 5: Subclass Determination and complement fixation assays. Autoantibody subclass was determined via dot blot and complement fixation was evaluated by IF. (A) Serial dilutions (1:4, 1:8, 1:16, 1:32) of supernatants from 293 EBNA HEK cells expressing sec180 was blotted onto a nitrocellulose membrane and cut into strips. Strips were incubated with a 1:50 dilution of sera followed by subclass specific secondaries. IgG1, known for high affinity for phagocytic cells and as a high complement activator, was the predominant subclass. Complement fixation was evaluated using IgG purified from a BP patient (A, positive control), or IgG purified from 3 individual PD sera (panels C- D), which were all negative. IgG1 in PD 1 IgG1 in PD 2 IgG1 in PD 3 IgG1 in BP control A. Over 1.5 million people in the United States are currently affected by Parkinson’s disease (PD). 1 PD is characterized by the loss of dopaminergic neurons in the substantia nigra. 2,3 Recent data indicates a strong epidemiologic link between PD and development of the autoimmune blistering disease, bullous pemphigoid (BP). PD patients are 2-4 times more likely to develop Bullous Pemphigoid (BP), a blistering disease characterized by autoantibodies to Collagen XVII (Col XVII), a hemidesmosomal protein involved in keratinocyte adhesion. 4,5 Col XVII is also expressed in neurons, where it is a cytoplasmic protein. 6 Our lab has shown that Col XVII autoantibodies are found in one third of PD patients without skin disease and these autoantibodies bind to tyrosine hydroxylase positive neurons in the substantia nigra. This binding is abrogated with pre-absorption of sera with recombinant Col XVII. Despite the strong association of BP and neurologic disease, the mechanism by which PD leads to a break in tolerance to Col XVII and results in autoimmunity is not understood. The purpose of this study was to determine if collagen XVII-specific autoantibodies in sera from Parkinson's patients also bind to the basement membrane zone of human skin. Additionally, these autoantibodies were characterized by immunoglobulin subtype and for their ability to fix complement. We hypothesize that sera from patients with PD who possess IgG reactivity to collagen XVII by immunoblot but do not have BP will not bind to the epidermal basement membrane zone or fix complement Fig. 3B: Indirect Immunofluorescence BMZ A.