Use of a Novel Probe to Demonstrate Granzyme B Activity in Sjogren’s Syndrome Salivary Glands

Kimberly Doering Maurer¹, Laura Gutierrez-Alamillo¹, Efstathia K. Kapsogeorgou², Athanasios G. Tzioufas³, Livia Casciola-Rosen⁴ and Antony Rosen⁴, ¹Medicine (Rheumatology), Johns Hopkins University School of Medicine, Baltimore, MD, ²Pathophysiology, School of Medicine, National University of Athens, Greece, Athens, Greece, ³Pathophysiology, School of Medicine, National University of Athens, Athens, Greece, ⁴Division of Rheumatology, Johns Hopkins University, Baltimore, MD

Meeting: 2012 ACR/ARHP Annual Meeting

Keywords: Sjogren's syndrome

Session Information

Title: Sjögren's Syndrome - Pathogenesis

Session Type: Abstract Submissions (ACR)

Background/Purpose: Salivary glands are a prominent target organ in Sjögren’s syndrome (SS), with patients having abnormal secretory function and inflammatory infiltration of these glands. Little is currently known about the mechanisms whereby the immune system contributes to ongoing tissue damage and dysfunction in this disease. IRF1 is one of the most efficiently cleaved granzyme B substrates ever defined. The purpose of this study was to define whether cytotoxic lymphocytes in the gland actively induce proteolysis in salivary gland epithelial cells in situ by using an antibody highly specific for granzyme B (GrB)-cleaved IRF-1 to probe for activity of the GrB pathway in vivo in SS salivary gland biopsies.

Methods: Immunohistochemical staining of salivary gland paraffin sections was used to assess the presence of CD8 infiltrates, and the extent of GrB staining. Lysates generated from frozen salivary gland biopsies were immunoblotted to detect CD8 and IRF1 expression. ³⁵S-methionine labeled IRF1 was generated by in vitro transcription and translation, and was used to demonstrate cleavability by GrB, and as a template for site-directed D²⁰⁴A mutagenesis to confirm the GrB cleavage site in IRF1. Using this information, an antibody detecting exclusively the N-terminal GrB cleaved IRF1 fragment (R6017) (and not the intact molecule) was generated. This antibody was carefully validated and used for immunohistochemistry and immunoblotting on salivary gland tissue and lysates obtained from SS patients.

Results: CD8 infiltrates were prominent in ~60% of SS salivary glands by immunoblotting and immunohistochemistry. IRF1 is expressed at high levels in SS salivary glands, but not control salivary glands. IRF1 is efficiently cleaved by GrB at D²⁰⁴. Using the antibody R6017 that is highly specific for the N-terminal fragment generated by GrB cleavage, we demonstrated the presence of GrB-induced fragments by immunoblotting and immunohistochemistry performed on SS patient but not control salivary gland tissue.

Conclusion: These studies provide the first direct in vivo evidence that the cytotoxic lymphocyte granule pathway is actively modifying salivary epithelial cells in patients with SS. Defining granzyme-induced effects on salivary epithelial function may provide important understanding of the mechanisms of salivary epithelial function in this disease, with therapeutic implications.

Disclosure:

K. Doering Maurer,
None;

L. Gutierrez-Alamillo,
None;

E. K. Kapsogeorgou,
None;

A. G. Tzioufas,
None;

L. Casciola-Rosen,
None;

A. Rosen,
None.

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