Molecular Analysis of the Human Autoantibody Response to α-Fodrin in Sjögren's Syndrome Reveals Novel Apoptosis-Induced Specificity

Abstract

Lymphocyte infiltration of salivary and lacrimal glands leading to diminished secretion and gland destruction as a result of apoptosis is thought to be pivotal in the pathogenesis of Sjögren's syndrome (SS). The cytoskeletal protein alpha-fodrin is cleaved during this apoptotic process, and a strong antibody (Ab) response is elicited to a 120-kd fragment of cleaved alpha-fodrin in the majority of SS patients, but generally not in other diseases in which apoptosis also occurs. Little is known about the anti-alpha-fodrin autoantibody response on a molecular level. To address this issue, IgG phage display libraries were generated from the bone marrow of two SS donors and a panel of anti-alpha-fodrin IgGs was isolated by selection on alpha-fodrin immunoblots. All of the human monoclonal Abs (hmAbs) reacted with a 150-kd fragment and not with the 120-kd fragment or intact alpha-fodrin, indicating that the epitope recognized became exposed after alpha-fodrin cleavage. Analysis of a large panel of SS patients (defined by the strict San Diego diagnostic criteria) showed that 25% of SS sera exhibited this 150-kd alpha-fodrin specificity. The hmAbs stained human cultured salivary acinar cells and the staining was redistributed to surface blebs during apoptosis. They also stained inflamed acinar/ductal epithelial cells in SS salivary tissue biopsies, and only partially co-localized with monoclonal Abs recognizing the full-length alpha-fodrin. Our study shows that in SS patients, neoepitopes on the 150-kd cleaved product of alpha-fodrin become exposed to the immune system, frequently eliciting anti-150-kd alpha-fodrin Abs in addition to the previously reported anti-120-kd Abs. The anti-150-kd alpha-fodrin hmAbs may serve as valuable reagents for the study of SS pathogenesis and diagnostic analyses of SS salivary gland tissue.

Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways

“…Alpha II-spectrin, besides being an important cytoskeletal protein, has been identified as a protein important to maintaining cell integrity and is found cleaved in the salivary glands cells of SS patients. Alpha II-spectrin has been proposed to be an autoantigen for Sjögren's syndrome [5,10,[45][46][47][48][49][50]; therefore, it is possible that the apoptotic cell death in the salivary gland cells of SS patients may somehow produce this autoantigen, but how this occurs is unclear.…”

“…The glands most affected in SS include the tear glands and the salivary glands. Histological studies indicate an increased infiltration of immune cells is present in the parotid and submandibular salivary gland epithelial cells of SS patients [9][10][11]. Activated immune cells secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma) and Fas ligand (FasL) and increased secretion of inflammatory cytokines has been associated with salivary gland cell death in SS patients [11][12][13][14][15].…”

“…The human salivary gland cell line, HSG, was isolated from an irradiated carcinoma of the submandibular gland of the human salivary gland epithelial cells [44]. HSG cells reproduce several properties of the SS disease process that occurs in vivo in patients [10,16,17,[57][58][59]. Exposure of HSG cells to TNF-alpha and IFN-gamma has been shown to inhibit cell growth and reduce cell number [18].…”

Discrimination between Sjögren's and Non-Sjögren's Sicca Syndrome by Sialoscintigraphy and Antibodies against α-Fodrin and Ro/La Autoantigens

“…8 The autoantigen, α-fodrin, has been postulated to be involved in the autoimmune responses leading to glandular destruction of lacrimal and salivary glands. 9 The presence of α-fodrin autoantigen has been found in 93% of primary SS patients, 10 and it has been reported that α-fodrin is a specific and sensitive marker for SS diagnosis.…”

NF-κB Controls Resistance of Human Salivary Gland (HSG) Cells to Apoptosis in an <i>in Vitro</i> Model of Sjogren’s Syndrome

“…Furthermore, salivary gland cells from patients with SS exhibit greater expression of Fas receptor (CD95), a death domain-containing member of the TNF receptor superfamily, which mediates apoptosis triggered by Fas ligand (FasL)-expressing lymphocytes [7]. Apoptosis in salivary tissue can be induced in vitro by Tumor Necrosis Factor (TNF): TNF, previously known as TNF-α, is a pleiotrophic cytokine produced by immune cells in response to infection, environmental challenges and/or other danger signals [8]- [10]. As an early pro-inflammatory cytokine, TNF stimulates production of various cytokines, chemokines, coagulation factors, and other inflammatory mediators, and up-regulates the expression of adhesion molecules [11].…”