Autoimmunity: Methods and Protocols

Field: Immunology and Microbiology (Life Sciences)

Edited by Andras Perl, 431 pp, Totowa, NJ, Humana Press, 2004.Autoimmunity affects approximately 10% of the population and is characterized by a breakdown in the homeostatic mechanisms that regulate autoreactive T and B lymphocytes. This dysregulation of self-recognition gives rise to a wide spectrum of autoimmune disorders that may involve multiple or single organ systems, which are clearly defined in the introductory chapter 1. This book is a part of the Methods in Molecular Medicine series. The remaining 21 chapters are divided into 2 general categories: “Part 1, Models of Pathogenesis in Human Autoimmune Disorders” (8 chapters) and “Part II, Animal Models of Autoimmunity” (13 chapters). The format for each chapter is similar and includes an introduction to the principles and importance of the techniques used, followed by a step-by-step protocol to perform assays, including reagents; cell types; experimental methods; and notes defining precautions, pitfalls, and resolutions. The methods, although specific for the model described, may be adapted to other experimental animal or cell culture models.Part I devotes 6 of its 8 chapters to the clinically heterogeneous autoimmune disorder, systemic lupus erythematosus (SLE). Chapter 2 describes whole genome search for SLE susceptibility genes, using linkage analysis and study of candidate gene. These methods have yielded only 1 susceptibility gene to date, namely, the programmed cell death 1 gene. This chapter does not contain detailed protocols. Systemic lupus erythematosus T lymphocytes express multiple abnormalities in signal transduction, which are described in the remaining 5 SLE-focused chapters. Chapter 3 explores potential abnormalities in T-cell signaling, including abnormal tyrosine phosphorylation of cellular proteins, intracellular calcium ion response, cytokine expression, and cell proliferation. Chapter 8 provides more detail in the evaluation of overexpression of type I interferons and interferon gene targets in SLE. The assessment of specific messenger RNA (mRNA) content by real-time polymerase chain reaction using 3 housekeeping genes for normalization is less reliable than the determination of specific proteins by Western blot or intracellular immunofluorescence staining. The difference in reliability is attributed to the variable stability of mRNA, potential absence of translation of mature in mRNA, and variability in housekeeping gene expression. Not mentioned is the lack of stabilization of mRNA at the time of phlebotomy, which may lead to overexpression or underexpression in viable lymphocytes before cellular and/or mRNA purification.1 The vital role T-cell receptor zeta-chain plays in receptor signaling is outlined in chapter 4. Various molecular intermediates of T-cell receptor zeta-chain exist and are altered in circulating T cells in patients with SLE compared to controls, including the lipid raft–associated form of T-cell receptor zeta-chains, which is increased in SLE. Protein kinase A isoenzyme activities are deficient, leading to impaired cAMP-dependent protein phosphorylation in SLE (chapter 5). Chapter 6 reviews the detection of dysfunctional apoptosis and mitochondrial activity in the lymphocytes of patients with SLE. Detection methods include lymphocyte activation, apoptosis assays, measuring changes in mitochondrial membrane potential during initiation of apoptosis using 3 dyes (DiOC6, TMRM, and JC-1), reactive oxygen intermediates, high-performance liquid chromatography analysis of oxidized/reduced forms of glutathione, and caspase enzyme activities. Another approach to screening for mitochondrial dysfunction involves monitoring the production of 14CO2 from [1-14C] lactate. The final step in this reaction is catalyzed by mitochondrial enzyme pyruvate dehydrogenase: [1-14C] pyruvate to 14CO2. This reaction is decreased in fibroblasts from patients with peroxisomal and mitochondrial disorders.2 The detection of the fate of autoimmune disease autoantigens during apoptosis is described in chapter 7. Transaldolase is the rate-limiting enzyme in the pentose phosphate pathway, which is necessary for myelination and an autoantigen in multiple sclerosis (chapter 9). A Western blot method is used to detect transaldolase autoantibodies and epitope mapping to define antigen-antibody binding requirements.Part II focuses on a great variety of animal models, primarily mice, used to study the pathogenesis of autoimmunity. A few examples include coxsackievirus B3–induced myocarditis, cardiac myosin–induced experimental autoimmune myocarditis, murine thyroglobulin–induced experimental autoimmune thyroiditis, and spontaneous experimental autoimmune thyroiditis in nonobese diabetic mice. H2h4 mice that do not develop diabetes (chapter 10), a nonobese diabetic mouse that develops autoimmune-mediated diabetes mellitus (chapter 11), cloned lines of T cells reactive for self-antigen from nonobese diabetic mice (chapter 12), murine SLE models (chapter 13), and a mouse model for collagen-induced arthritis (chapter 16) are discussed. Also discussed is proteoglycan aggrecan–induced arthritis (chapter 17), as well as 2 mouse models of multiple sclerosis in chapter 18 (experimental autoimmune encephalomyelitis and Theiler virus–induced demyelinating disease).Experimental autoimmune encephalomyelitis mouse model for multiple sclerosis is again covered in chapter 19, while systemic sclerosis in mice after subcutaneous bleomycin infection is discussed in chapter 20. Rodent models of experimental autoimmune uveitis (chapter 21) and C57 BL/6 mouse model for vitiligo (chapter 22) are also discussed.This how-to-do-it compendium of methods to explore the pathogenesis of autoimmune disease in humans and animal models is well written. The protocols are easy to follow but may require additional reading for the novice. A wide variety of autoimmune disorders are discussed, especially in part II. This volume would be useful for the graduate student, experimental pathologist, or basic science investigator interested in evaluating novel methods for experimental design in the field of autoimmunity.