Research ArticleAutoimmunity

Laminin 511 is a target antigen in autoimmune pancreatitis

See allHide authors and affiliations

Science Translational Medicine  08 Aug 2018:
Vol. 10, Issue 453, eaaq0997
DOI: 10.1126/scitranslmed.aaq0997
  • Fig. 1 Detection by ELISA of laminin 511-E8 antibodies in AIP patient sera.

    Serum antibodies against laminin 511-E8, laminin 511-FL, laminin 521-E8, laminin 521-FL, collagen IV, and fibronectin were quantified by ELISA in a validation cohort. Forty-one AIP patients and 112 controls (20 normal and 92 diseased) were examined. The cutoff value (OD) was defined as the mean + 3 SDs of the control sera. The dashed line indicates the cutoff value.

  • Fig. 2 Colocalization of laminin and IgG in AIP patient pancreatic tissue.

    Immunohistochemical studies of pancreatic tissues from AIP patients. (A) Top: Hematoxylin and eosin (H&E) staining (left) and immunohistochemical staining for IgG (middle) and laminin (right) in affected tissue sections from an AIP patient (patient numbers: AIP52 to AIP56 in table S3). Scale bars, 200 μm. Bottom: High-power magnification of the upper panels. Scale bars, 20 μm. (B) Immunofluorescence staining of the affected acini in the AIP patient. Top: Staining for IgG (green), laminin (red), and a merged image. Bottom: Immunofluorescence staining for IgG1 (green), laminin (red), and a merged image. Scale bars, 20 μm. (C) Immunofluorescence staining of the unaffected acini in the AIP patient. Scale bars, 20 μm. Left lower white boxes are magnified images of the dashed line boxes. Sections were stained with polyclonal anti-mouse/human laminin antibody produced against the protein purified from the basement membrane of Engelbreth-Holm-Swarm sarcoma, and the antibody reacts with several laminin family proteins of mouse and human, including human laminin 511-E8, but does not react with other ECM proteins examined.

  • Fig. 3 Colocalization of AIP patient IgG and laminin in mouse pancreatic tissue by passive transfer of patient IgG.

    Immunofluorescence studies were performed 12 hours after subcutaneous injection of control IgG or AIP patient IgG. (A) Immunofluorescence staining for human IgG in mouse pancreatic tissue sections. Top right three panels show staining for patient IgG (green), amylase (red), and a merged image. Bottom right three panels show staining for patient IgG (green), laminin (red), and a merged image. Scale bars, 20 μm. (B) Immunofluorescence staining for human IgG1 and laminin in mouse pancreas. Staining for a patient’s IgG1 (green), laminin (red), and a merged image are shown. Scale bars, 20 μm. (C) When AIP patient IgG was preincubated with laminin 511-E8 or 511-FL, IgG staining with laminin (left panel) was abolished only by preincubation with laminin 511-E8 (middle panel), but not by preincubation with laminin 511-FL (right panel). Scale bars, 20 μm. Representative photos are shown.

  • Fig. 4 Induction of AIP-like lesions in mice by immunization with human laminin 511-E8.

    BALB/c mice were immunized with human laminin 511-E8 (right panels) or ovalbumin (left panels) once a month for 3 months. H&E staining (A); immunohistochemical staining for CD3, CD45R, CD138, and IgG1 (B); and elastica van Gieson staining (C) of mouse pancreatic tissue sections obtained 28 days after the last immunization are shown. Among various organs (brain, salivary gland, thyroid, heart, lung, gallbladder, bile duct, pancreas, kidney, intestine, bladder, prostate, aorta, skeletal muscles, and skin), only the pancreas and salivary gland (fig. S9B) exhibited injury in all mice injected with laminin 511-E8 (n = 5), whereas none of the ovalbumin mice (n = 5) had any lesions (five of five versus zero of five, P < 0.005). Subepithelial space enlargement in bile and pancreatic duct with no epithelial changes, typical features of AIP, were also observed (A, bottom panels, black arrows). IgG1, a counterpart of human IgG4, was also detected (B, bottom right panel). Scale bars, 50 μm (A) and 20 μm (B).

  • Fig. 5 Detection of the integrin α6β1 antibody in a limited number of AIP patients negative for the anti–laminin 511-E8 antibody.

    The ELISA results of the 51 AIP patients and 122 controls are shown. The cutoff value (OD) was defined as the mean + 3 SDs of the control sera. The dashed line indicates the cutoff value.

  • Table 1 Clinical differences between AIP patients positive and negative for anti–laminin 511-E8 antibodies.

    Malignancy includes those diagnosed at the same time of or after AIP diagnosis. Hypocomplement includes low C3, C4, or CH50. Allergy includes asthma, atopic dermatitis, drug allergy, food allergy, and contrast media hypersensitivity. Values are medians for age, serum IgG, and serum IgG4.

    Anti–laminin 511-E8P
    PositiveNegative
    No. of AIP patients2625
    Sex (male/female)19/721/40.34
    Age67630.53
    Serum IgG (mg/dl)212020400.35
    Serum IgG4 (mg/dl)4474080.45
    Diffuse/focal*22/416/90.091
    Head involvement (%)*2 (8)18 (72)<0.001
    Malignancy (%)*0 (0)8 (32)0.0017
    Diabetes mellitus (%)*11 (44)10 (40)0.87
    Hypocomplement (%)*3/18 (12)5/21 (23)0.41
    Allergy (%)*3 (12)12 (48)0.0043

    *The number of patients is shown.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/453/eaaq0997/DC1

      Fig. S1. Schematic representations of the laminin full-length (laminin-FL) and laminin E8 isoforms.

      Fig. S2. Screening of antigens in AIP.

      Fig. S3. Detection of C-terminal fragments of α, β, and γ chains of laminin 511 in mouse and human pancreas by Western blot analysis.

      Fig. S4. Detection by ELISA of antibody against human laminin 511-E8 in the sera of AIP patients.

      Fig. S5. IgA, IgM, and IgE antibodies against human laminin 511-E8 in the sera of AIP patients and titration of sera against laminin 511-E8.

      Fig. S6. No reaction of patient sera with laminin 511-E8 by Western blot analysis.

      Fig. S7. Expression and colocalization of laminin 511 and each chain of laminin in normal mouse and human pancreatic tissue.

      Fig. S8. Quantification of the merged area in the pancreatic tissue of AIP patients and mouse pancreas of passive transfer model.

      Fig. S9. Induction of antibody against human laminin 511-E8 and salivary lesions by immunization with human laminin 511-E8.

      Fig. S10. Relationship between pancreatic image and positivity for the laminin 511-E8 antibody.

      Fig. S11. Changes in antibody titers against laminin 511-E8 by steroid treatment.

      Table S1. Clinical information on all patients and controls.

      Table S2. Clinical differences among AIP patients with anti–laminin 511-E8 antibody, anti–integrin α6β1 antibody, or neither antibody.

      Table S3. Clinical information on five AIP patients and controls whose pancreatic tissues were used in experiments.

      Table S4. Antigens used for ELISA in this study.

      Table S5. Primary data.

    • The PDF file includes:

      • Fig. S1. Schematic representations of the laminin full-length (laminin-FL) and laminin E8 isoforms.
      • Fig. S2. Screening of antigens in AIP.
      • Fig. S3. Detection of C-terminal fragments of α, β, and γ chains of laminin 511 in mouse and human pancreas by Western blot analysis.
      • Fig. S4. Detection by ELISA of antibody against human laminin 511-E8 in the sera of AIP patients.
      • Fig. S5. IgA, IgM, and IgE antibodies against human laminin 511-E8 in the sera of AIP patients and titration of sera against laminin 511-E8.
      • Fig. S6. No reaction of patient sera with laminin 511-E8 by Western blot analysis.
      • Fig. S7. Expression and colocalization of laminin 511 and each chain of laminin in normal mouse and human pancreatic tissue.
      • Fig. S8. Quantification of the merged area in the pancreatic tissue of AIP patients and mouse pancreas of passive transfer model.
      • Fig. S9. Induction of antibody against human laminin 511-E8 and salivary lesions by immunization with human laminin 511-E8.
      • Fig. S10. Relationship between pancreatic image and positivity for the laminin 511-E8 antibody.
      • Fig. S11. Changes in antibody titers against laminin 511-E8 by steroid treatment.
      • Table S1. Clinical information on all patients and controls.
      • Table S2. Clinical differences among AIP patients with anti–laminin 511-E8 antibody, anti–integrin α6β1 antibody, or neither antibody.
      • Table S3. Clinical information on five AIP patients and controls whose pancreatic tissues were used in experiments.
      • Table S4. Antigens used for ELISA in this study.

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Table S5 (Micorsoft Excel format). Primary data.

    Navigate This Article