Autoantibody Profiling Using Antigen Arrays
Autoimmune disease affects 3% of the U.S. population, and likely a similar percentage of the industrialized world population (1). Although we have made remarkable progress towards understanding immune function over the past 4 decades in terms of the role of the major histocompatibility complex (MHC) and the nature of lymphocyte antigen receptors that confer specificity to autoimmune responses, our understanding of the underlying dysregulation and specificity of autoimmune responses remains limited. For certain autoimmune diseases, including multiple sclerosis (MS) and systemic lupus erythematosus (SLE), candidate autoantigens have been identified but their exact role in the initiation, perpetuation, and pathophysiology are not well understood. For other autoimmune diseases, including rheumatoid arthritis (RA) and psoriasis, the targeted autoantigens remain unidentified despite extensive experimental efforts. Array and other multiplex screening technologies represent powerful tools to study the pathophysiology and specificity of autoimmune responses.
The advent of DNA microarray technology during the last decade has led to an explosion of studies aimed at identifying novel messenger RNA transcripts, or patterns of transcripts, that are transcriptionally up- or down-regulated in association with a particular disease or phenotype. As the availability and costs of such 'DNA chips' improve, it is anticipated that transcriptional profiling will gain even greater prominence in autoimmune disease research. 'Spotted' DNA microarrays are now available at many university and industry laboratories, and are providing a wealth of information regarding the underlying pathophysiology of autoimmune disease (2). However, use of RNA transcipt profiling has important limitations and is likely unable to provide a comprehensive understanding of autoimmune processes necessary to develop next-generation therapeutics.
RNA transcript profiling alone is an inadequate method for studying human autoimmune disease for several reasons. First, diseases are manifest not at the level of RNA transcription, but rather at the level of the protein. Second, there exists a nonpredictive correlation between RNA expression and protein expression and function (3, 4). Messenger RNA undergoes a variety of processing events that can profoundly effect cell phenotype, yet are not revealed in current transcriptional profiles. For example, messenger RNA encoding certain apoptosis regulatory molecules exists in 2 or more alternative splice forms encoding proteins with opposing functions (e.g., proapoptotic isoforms such as bcl-xS and protective isoforms such as bcl-xL) (5). Translation of mRNA into protein is also regulated by translational regulatory elements such as 3' mRNA AU-rich elements and by addition of polyA tails of varying lengths (6-8). Third, protein function can be regulated by post-translational modifications by enzymes such as kinases or proteases. Finally, autoimmune responses are regulated by autoantigen-specific T and B lymphocytes expressing distinct and heterogeneous antigen receptors not easily examined by transcriptional profiling. Many of these limitations can be circumvented by direct study of the expression and function of proteins encoded by these RNA transcripts. The study of such proteins and protein-protein interactions is termed 'proteomics' (9). With our entrance into the 'post-genomics era' it is essential to develop novel tools with which protein expression and protein-protein interactions can be explored.
Several groups, including MacBeath and Schreiber at Harvard (36) and Haab and Brown at Stanford (37), developed methods utilizing such robotic arrayers to deposit proteins onto microscope slides to fabricate high-density protein arrays. To date, comprehensive autoantigen microarrays have not yet been described that are capable of fluorescent detection of human autoantibodies present in biological fluids such as serum, cerebrospinal fluid (CSF), or synovial fluid. We therefore developed and refined protein microarray technology to study the specificity of the autoantibody response in murine and human autoimmune diseases (Robinson et al., 2002). Using a modified protocol from that recently described by Haab et al. and MacBeath and Schreiber (36, 37), we used a robotic arrayer to attach peptides, proteins, nucleic acids, and protein complexes to distinct and addressable locations on microscope slides. At each antigen feature approximately 1 nanoliter of antigen solution is deposited producing a relatively uniform feature measuring 150 (m in diameter. Individual arrays are incubated with serum derived from autoimmune disease and control patients, washed, and incubated with secondary antibodies covalently-conjugated to spectrally-resolvable fluorescent labels (such as Cy-3 or Cy-5). Alternatively, comparative analysis can be performed by incubating arrays simulataneously with a reference and disease serum sample, each directly labeled with a distinct spectrally-resolvable fluorochrome as described by Haab et al. (37). The slides are analyzed using a fluorescence-based digital scanning system. Antigen microarrays employ simple protocols and basic spotted DNA array equipment available at many academic medical centers and industry laboratories. Detailed protocols to fabricate and conduct antigen microarray studies can be found on the world wide web at http://www.stanford.edu/groups/antigenarrays and http://cmgm.stanford.edu/pbrown. The spotted array studies are an active component of the Utz lab's efforts. Autoantigen array technology and their use to guide selection of antigen-specific tolerizing therapies was conceived of by Bill Robinson, who works jointly in the Utz and Steinman labs on this and several other projects.
REFERENCES:
1. Jacobson DL, Gange SJ, Rose NR, Graham NM. Epidemiology and estimated
population burden of selected autoimmune diseases in the United States.
Clin Immunol Immunopathol 1997;84(3):223-43.
2. Khan J, Bittner M, Chen Y, Meltzer P, Trent J. DNA microarray technology:
the anticipated impact on the study of human disease. Biochim Biophy Acta
1999;1423:M17-M28.
3. Futcher B, Latter G, Monardo P, McLaughlin C, Garrels J. A sampling
of the yeast proteome. Mol Cell Biol 1999;19:7357-7368.
4. Varshavsky A. The N-end rule: functions, mysteries, uses. Proc Natl
Acad Sci, USA 1996;93:12142-12149.
5. Jiang Z, Wu J. Alternative splicing and apoptosis. Proc Soc Exp Biol
Med 1999;220:64-72.
6. Jackson R, Standart N. Do the poly(A) tail and 3' untranslated region
control mRNA translation? Cell 1990;62:15-24.
7. Sheets M, Fox C, Hunt T, Vande Woude G, Wickens M. The 3'-untranslated
regions of c-mos and cyclin mRNAs stimulate translation by regulating cytoplasmic
polyadenylation. Genes Dev 1994;8:926-938.
8. Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F, Kollias G.
Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU- rich
elements: implications for joint and gut-associated immunopathologies. Immunity
1999;10(3):387-98.
9. Pandey A, Mann M. Proteomics to study genes and genomes. Nature 2000;405:837-846.
10. Rohlff C. Proteomics in molecular medicine: applications in central
nervous system disorders. Electrophoresis 2000;21:1227-1234.
11. Borrebaeck C. Antibodies in diagnostics - from immunoassays to protein
chips. Immunology Today 2000;21:379-382.
12. Emili A, Cagney G. Large-scale functional analysis using peptide
or protein arrays. Nature Biotech 2000;18:393-397.
13. Walter G, Büssow K, Cahill D, Lueking A, Lehrach H. Protein arrays
for gene expression and molecular interaction screening. Curr Opin Microbiol
2000;3:298-302.
14. Gygi S, Corthals G, Zhang Y, Rochon Y, Aebersold R. Evaluation of
two-dimensional gel electrophoresis-based proteome analysis technology.
Proc Natl Acad Sci, USA 2000;97:9390-9395.
15. Fields S, Song O. A novel genetic system to detect protein-protein
interactions. Nature 1989;340(6230):245-6.
16. Uetz P, al e, Fields S. A comprehensive analysis of protein-protein
interactions in Saccaromyces cerevisiae. Nature 2000;403:623-627.
17. von Mühlen CA, Tan EM. Autoantibodies in the diagnosis of systemic
rheumatic diseases. Semin Arthritis Rheum 1995;24(5):323-358.
18. Meinhart CD, Wereley ST, Santiago JG. "Diagnostic Techniques for
Microfluidics Research,". Developments in Laser Techniques and Applications
to Fluid Mechanics, R. J. Adrian et al. (Eds.), Springer-Verlag, Berlin,
1999. 2000.
19. Santiago JG, Wereley ST, C.D. Meinhart. A Velocimetry Technique for
Microfabricated Fluidic Systems. Journal of Microelectromechanical Systems
2000;submitted.
20. Kawaoka J, Gomez F. Use of mobility ratios to estimate binding constants
of ligands to proteins in affinity capillary electrophoresis. J Chromatography
1998;715:203-210.
21. Beebe D, Moore J, Yu Q, Liu R, Kraft M, Jo B-H, et al. Microfluidic
tectonics: a comprehensive construction platform for microfluidics systems.
Proc Natl Acad Sci, USA 2000;97:13488-13493.
22. Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JR, Jr. Advanced
multiplexed analysis with the FlowMetrix system. Clin Chem 1997;43(9):1749-56.
23. Walt DR. Techview: molecular biology. Bead-based fiber-optic arrays.
Science 2000;287(5452):451-2.
24. del Rincon I, Zeidel M, Rey E, Harley J, James J, Fischbach M, et
al. Delineation of human systemic lupus erythematosus anti-smith antibody
response using phage display combinatorial libraries. J Immunol 2000;165:7011-7016.
25. Ekins RP. Multi-analyte immunoassay. J Pharm Biomed Anal 1989;7(2):155-68.
26. Ekins R, Chu F. Multianalyte microspot immunoassay. The microanalytical
'compact disk' of the future. Ann Biol Clin 1992;50(5):337-53.
27. Ekins R, Chu F, Micallef J. High specific activity chemiluminescent
and fluorescent markers: their potential application to high sensitivity
and 'multi-analyte' immunoassays. J Biolumin Chemilumin 1989;4(1):59-78.
28. Mendoza L, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers
M. High-throughput microarray -based enzyme-linked immunosorbant assay (ELISA).
Biotechniques 1999;27:778-788.
29. Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed,
spatially addressable parallel chemical synthesis. Science 1991;251(4995):767-73.
30. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR,
et al. Maskless fabrication of light-directed oligonucleotide microarrays
using a digital micromirror array. Nat Biotechnol 1999;17(10):974-8.
31. Blanchard AP, Friend SH. Cheap DNA arrays-it's not all smoke and
mirrors. Nat Biotechnol 1999;17(10):953.
32. Ge H. UPA, a universal protein array system for quantitative detection
of protein-protein, protein-DNA, protein-RNA, and protein-ligand interactions.
Nucleic Acids Res 2000;28.
33. Meheus L, van Venrooij WJ, Wiik A, Charles PJ, Tzioufas AG, Meyer
O, et al. Multicenter validation of recombinant, natural and synthetic antigens
used in a single multiparameter assay for the detection of specific anti-nuclear
autoantibodies in connective tissue disorders. Clin Exp Rheumatol 1999;17(2):205-14.
34. Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of
gene expression patterns with a complementary DNA microarray. Science 1995;270(5235):467-70.
35. Haab BB, Dunham MJ, Brown PO. Protein microarrays for highly parallel
detection and quantitation of specific proteins and antibodies in complex
solutions. Genome Biol 2001;2(2).
36. MacBeath G, Schreiber S. Printing proteins as microarrays for high-throughput
function determination. Science 2000;289:1760-1763.
37. Haab B, Dunham M, Brown P. Protein microarrays for highly parallel
detection and quantitation of specific proteins and antibodies in complex
solutions. Genome Biology 2001;In Press.
38. Joos T, Schrenk M, Hopfl P, Kroger K, Chowdhury U, Stoll D, et al.
A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics.
Electrophoresis 2000;21:2641-2650.
39. Casciola-Rosen LA, Miller DK, Anhalt GJ, Rosen A. Specific cleavage
of the 70 kDa protein component of the U1 small nuclear riboprotein is a
characteristic biochemical feature of apoptotic cell death. J Biol Chem
1994;269(49):30757-30760.
40. Casciola-Rosen L, Andrade F, Ulanet D, Wong W, Rosen A. Cleavage
by granzyme B is strongly predictive of autoantigen status. Implications
for initiation of autoimmunity. J Exp Med 1999;190:815-26.
41. Utz PJ, Gensler TJ, Anderson P. Death, autoantigen modifications,
and tolerance. Arthritis Res 2000;2(2):101-14.
42. Utz P, Anderson P. Posttranslational modifications, apoptosis, and
bypass of tolerance to autoantigens. Arthritis Rheum 1998; 41:1152-1160.
43. Venter J, al e. The sequence of the human genome. Science 2001;291:1304-1351.
44. Lueking A, Horn M, Eickhoff H, Bussow K, Lehrach H, Walter G. Protein
microarrays for gene expression and antibody screening. Anal Biochem 1999;270:103-111.
45. Bussow K, Cahill D, Nietfeld W, Bancroft D, Scherzinger E, Lehrach
H, et al. A method for global protein expression and antibody screening
on high-density filters of an arrayed cDNA library. Nucleic Acids Res 1998.
46. Craft J, Fatenejad S. Self antigens and epitope spreading in systemic
autoimmunity. Arthritis Rheum 1997;40:1374-1382.
47. James J, Harley J. B-cell epitope spreading in autoimmunity. Immunology
Reviews 1998;164:185-200.
48. Abbas A, Murphy K, Sher A. Functional diversity of helper T lymphocytes.
Nature 1996;383:789-793.
49. Liblau R, Singer S, McDevitt H. Th1 and Th2 CD4+ T cells in the pathogenesis
of organ-specific autoimmune diseases. Immunol Today 1995;16:34-38.
50. Harris D, Haynes L, Sayles P, Duso D, Eaton S, Lepak N, et al. Reciprocal
regulation of polarized cytokine production by effector B and T cells. Nature
Immunol 2000;1:475-482.
51. Whitney L, Becker K, Tresser N, Caballero-Ramos C, Munson P, Prabhu
V, et al. Analysis of gene expression in multiple sclerosis lesions using
cDNA microarrays. Ann Neurol 1999;46:425-428.
52. Zanders E, Goulden M, Kennedy T, Kempsell K. Analysis of immune system
gene expression in small rheumatoid arthritis biopsies using a combination
of subtractive hybridization and high-density cDNA arrays. J Immunol Methods
2000;233:131-140.
53. Ungethum U, Haupl T, Zacher J, Gursche A, Forster P, Reutermann P,
et al. Patterns of differentially expressed genes in synovial tissue from
RA and OA patients and from normal joints. Arth Res 2001;3:A16.
54. Weiner HL, Friedman A, Miller A, Khoury SJ, al-Sabbagh A, Santos
L, et al. Oral tolerance: immunologic mechanisms and treatment of animal
and human organ-specific autoimmune diseases by oral administration of autoantigens.
Annu Rev Immunol 1994;12:809-37.
55. Gaur A, Wiers B, Liu A, Rothbard J, Fathman CG. Amelioration of autoimmune
encephalomyelitis by myelin basic protein synthetic peptide-induced anergy.
Science 1992;258(5087):1491-4.
56. Warren KG, Catz I, Wucherpfennig KW. Tolerance induction to myelin
basic protein by intravenous synthetic peptides containing epitope P85 VVHFFKNIVTP96
in chronic progressive multiple sclerosis. J Neurol Sci 1997;152(1):31-8.
57. Brocke S, Gijbels K, Allegretta M, Ferber I, Piercy C, Blankenstein
T, et al. Treatment of experimental encephalomyelitis with a peptide analogue
of myelin basic protein. Nature 1996;379(6563):343-6.
58. Critchfield JM, Racke MK, Zuniga-Pflucker JC, Cannella B, Raine CS,
Goverman J, et al. T cell deletion in high antigen dose therapy of autoimmune
encephalomyelitis. Science 1994;263(5150):1139-43.
59. Furie RA, Cash JM, Cronin ME, Katz RS, Weisman MH, Aranow C, et al.
Treatment of systemic lupus erythematosus with LJP 394. J Rheumatol 2001;28(2):257-65.
60. Ruiz P, Garren H, Urbanek-Ruiz I, Hirschberg D, Nguyen L, Karpuj
M, et al. Suppressive immunization with DNA encoding a self-protein prevents
autoimmune disease: modulation of T cell costimulation. J Immunol 1999;162:3336-3341.
61. Garren H, Steinman L. DNA vaccination in the treatment of autoimmune
disease. Curr Dir Autoimmun 2000;2:203-16.
62. Utz P, Hottelet M, Schur P, Anderson P. Proteins phosphorylated during
stress-induced apoptosis are common targets for autoantibody production
in patients with systemic lupus erythematosus. J Exp Med 1997;185:843-854.
