Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays (original) (raw)
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Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays
Abstract
Nephrophilic autoantibodies dominate the seroprofile in lupus, but their fine specificities remain ill defined. We constructed a multiplexed proteome microarray bearing about 30 antigens known to be expressed in the glomerular milieu and used it to study serum autoantibodies in lupus. Compared with normal serum, serum from B6.Sle1.lpr lupus mice (C57BL/6 mice homozygous for the NZM2410/NZW allele of Sle1 as well as the FASlpr defect) exhibited high levels of IgG and IgM antiglomerular as well as anti–double-stranded DNA/chromatin Abs and variable levels of Abs to α-actinin, aggrecan, collagen, entactin, fibrinogen, hemocyanin, heparan sulphate, laminin, myosin, proteoglycans, and histones. The use of these glomerular proteome arrays also revealed 5 distinct clusters of IgG autoreactivity in the sera of lupus patients. Whereas 2 of these IgG reactivity clusters (DNA/chromatin/glomeruli and laminin/myosin/Matrigel/vimentin/heparan sulphate) showed association with disease activity, the other 3 reactivity clusters (histones, vitronectin/collagen/chondroitin sulphate, and entactin/fibrinogen/hyaluronic acid) did not. Human lupus sera also displayed 2 distinct IgM autoantibody clusters, one reactive to DNA and the other apparently polyreactive. Interestingly, the presence of IgM polyreactivity in patient sera was associated with reduced disease severity. Hence, the glomerular proteome array promises to be a powerful analytical tool for uncovering novel autoantibody disease associations and for distinguishing patients at high risk for end-organ disease.
Authors
Quan Li Zhen, Chun Xie, Tianfu Wu, Meggan Mackay, Cynthia Aranow, Chaim Putterman, Chandra Mohan
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letter about Zhen et al.
Submitter: Mart Mannik | mannik@u.washington.edu
University of Washington
Published March 8, 2006
To the Editors:
In the December issue of the Journal of Clinical Investigation the article by Zhen et al (1) the authors suggest that the glomerular proteome assay promises to be a powerful analytical tool in the study of lupus renal disease. Several issues need to be considered prior to achieving this promise.
First, serums from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis and other conditions are notorious for nonspecific binding to wells in enzyme-linked immunoassays and therefore for each serum a blank, consisting of the presence of the blocking agent and no antigen, should be included and the readout should be subtracted from the antigen coated and blocked well readout. The same appears to be true in the proteome array slides used by Zhen and colleagues. In Figure 5 several serums from SLE patients are shown in red, indicating high level of IgG binding, on spots with bovine serum albumin and on spots with ovalbumin, consistent with nonspecific binding. Bovine serum albumin was used as a blocking agent on all slides. The normalized fluorescence intensity for bovine serum albumin for each serum should have been subtracted from the readout for each antigen for each serum prior to further analysis of the data.
Second, the use of heat map diagrams for the analysis of antibody normalized fluorescence units is of questionable value. I:n the heat map analysis each row in the assay , including serums from controls, patients with SLE, and patients with rheumatoid arthritis, the mean for normalized fluorescence intensity units is calculated. Fluorescence intensities that were higher than the mean, were colored red, those below the mean were colored green, and values close to the mean were black. The mean of the fluorescence intensities is influenced by the number of normal controls as well as the number of very high levels of antibodies, thus not indicating positive and negative tests for the presence of antibodies. For clinicians the useful information is the finding that antibodies to an antigen are absent or present and the degree of positivity. It would be better to determine the fluorescence intensity for an adequate number of normal serums and then color code the test serums that are positive, e. g. two standard deviations above the mean for the control serums.
Third, it is useful to recall that all protein-protein interactions are not antigen-antibody interactions. Charge-charge interactions are important in binding of cationic proteins to the negatively charged glomerular basement membrane in vivo and in vitro. Aggregated IgG and immune complexes bind readily to histones and therefore some of the binding of IgG in SLE serums results from the presence of immune complexes and not from antibodies to histones (2, 3, 4). Furthermore, IgG may bind to vimentin via the Fc fragment of IgG (5). These are examples of some molecular interactions that can not be construed as antigen-antibody interactions,
Thus, modifications of the proteome arrays and the analysis of the readouts are needed before clinically useful data are obtained.
References
1. Zhen Q. L., Xie C., Wu T., Mackay M. et al. 2005. Identification of autoantibody clusters that best predict lupus disease activity using glomerular proteome arrays. J. Clin. Invvest. 115:3428- 3439.
2. Costa O., Marion C., Monier J. C. Roux B. 1984. Non-specific binding of heat-aggregated IgG to histone detected by ELISA. J. Immunol. Methods. 74:283-291.
3. Gussin H. A. E., Tselentis H. N., Teodorescu M. 2000. Noncognate binding to histone of IgG from patients with idiopathic systemic lupus erythematosus. Clin. Immunol. 96:150-161. 4. Mannik M.,Merrill C. E., Stamps L. D., Wener M. H. 2003. Multiple autoantibodies form the glomerular immune deposits in patients with systemic lupus erythematosus. J. Rheumatol. 30:1495-1504.
5. Hansson G. K., Starkebaum G.A., Benditt E. P., Schwartz S.M. 1984. Fc-mediated binding of IgG to vimentin-type intermediate filaments in vascular endothelial cells. Proc. Natl. Acad. Sci. USA. 81:3103-3107.