Does the cellular localization of antigens in or on apoptotic blebs influence the pathogenicity or benefit of cognate antibodies Comment on the article by Dieud┬й et al.код для вставкиСкачать
2080 A, Takei S, Sadeghi S, Stout A, Shaham B, Bernstein B, et al. Etanercept therapy in children with treatment-resistant uveitis. Arthritis Rheum 2001;44:1411–5). In that article we reported that etanercept injected subcutaneously twice weekly had a beneficial effect in 10 children (18 eyes) with treatmentresistant chronic uveitis. Within 3 months, 10 of 16 affected eyes (63%; P ⫽ 0.017) showed a rapid decrease in anterior chamber cell density, with remission achieved in 4 eyes. Among children with a visual acuity of ⬍20/25, 4 of 10 eyes (40%) improved. An exacerbation of uveitis during treatment with etanercept occurred in only 1 child (1 of 14 eyes) (7%). Other ocular outcome parameters, such as intraoccular pressure, synechiae formation, and lens clarity, remained unchanged. An increase in the dosage of etanercept to an average of 1.1 mg/kg after 3 months in 7 children failed to produce further improvement after 6 months of treatment. Since the completion of our original data accumulation, 2 years have passed. In the meantime, multiple studies describing the use of tumor necrosis factor antagonists in uveitis treatment have been published. Of particular interest are the results of a small double-blind placebo-controlled trial of etanercept in the treatment of pediatric uveitis, reported as an abstract at the 2002 American College of Rheumatology Annual Scientific Meeting (Smith JA, Smith S, Whitcup SM, Suhler E, Clarke G, Thompson D, et al. The treatment of JRA-associated uveitis with etanercept [abstract]. Arthritis Rheum 2002;46 Suppl 9:S482). In that study 12 children (mean age 11 years) were treated with either etanercept 0.4 mg/kg (maximum dosage 25 mg twice weekly) or placebo. After 6 months the authors were unable to detect any improvement in anterior chamber cell density in either patient group. In contrast, the beneficial effect of the treatment has been maintained in a majority of our patients. Of the 10 children originally enrolled, 2 withdrew within the first 6 months for reasons unrelated to the study drug. Both children had initially improved, and had flares after etanercept was discontinued. Four children did not respond to treatment, and 3 of them discontinued etanercept within 12 months. One child continues to have uncontrolled uveitis but still takes etanercept for control of his arthritis. Of the 4 children with sustained improvement, 1 girl continued to show improvement for 18 months, and her vision normalized. The family has since moved and the child was lost to followup. One girl who initially had a favorable response discontinued etanercept after 14 months of treatment when she experienced a grand mal seizure. Results of a diagnostic evaluation, including magnetic resonance imaging of the brain, lumbar puncture, and electroencephalography, were inconclusive for demyelinating disease. She was diagnosed as having benign childhood epilepsy (rolandic seizures) and started on gabapentin treatment. One month after discontinuation of etanercept she experienced a severe uveitis flare in both eyes. Etanercept was restarted 3 months later, and the uveitis again improved. She still has occasional mild seizure activity, while the uveitis in her left eye remains fully controlled and her right eye has minor inflammation. One girl continued to show improvement and had minimal uveitis but chose to discontinue etanercept after 22 months. She experienced a significant disease flare 2 months later. She was initially unable to restart etanercept due to a worldwide shortage of the drug. However, she was able to resume etanercept therapy after 15 months, LETTERS and her uveitis has improved significantly ever since. Lastly, one girl continues to improve, currently has no active uveitis, and her vision has significantly improved. In summary, in 6 of 10 children enrolled in this small, prospective open-label trial, etanercept had a beneficial effect for at least 3 months, and 4 children exhibited sustained benefit for at least 1 year. Etanercept may represent a viable alternative for children with active uveitis who have been treated unsuccessfully with traditional disease-modifying antirheumatic drugs. Andreas Reiff, MD University of Southern California Childrens Hospital Los Angeles Los Angeles, CA DOI 10.1002/art.11139 Does the cellular localization of antigens in or on apoptotic blebs influence the pathogenicity or benefit of cognate antibodies? Comment on the article by Dieudé et al To the Editor: Given the flood of attention directed toward understanding the affector arm in autoimmune diseases, studies that address the effector arm to study the pathogenicity of the perpetrator are of great interest. In certain circumstances the effects of an autoantibody may even be beneficial. This was in fact the situation being studied by Dieudé et al, who sought the mechanism by which autoantibodies to nuclear lamin B1 abrogate the strong prothrombotic risk associated with the lupus anticoagulant (1). The authors tested the hypothesis that circulating anti–lamin B1 antibodies block the procoagulant effect of apoptotic blebs by binding to lamin B1 displayed at the external surface of the blebs. However, by both biochemical and morphologic criteria, their extensive studies convincingly negated this notion. Lamin B1, in contrast to SSB/La, is not present on the surface of the apoptotic blebs but rather remains buried within the bleb, thereby being inaccessible to external anti–lamin B1 antibodies. Although these findings were considered negative in one context, they are indeed informative when considering another context. As discussed by the authors, although apoptotic blebs serve as an important target for certain autoantibodies, it is now apparent that this is not generalizable to all autoantigens. With regard to the pathogenicity of anti-SSA/Ro and anti-SSB/La in the development of congenital heart block, unambiguous demonstration of antibody binding to the surface of apoptotic cardiomyocytes was a critical link that provided a plausible explanation of how antibodies were accessible to otherwise sequestered intracellular antigens (2). If intracellular trafficking of sequestered nuclear antigens to the membrane surface is not universally applicable to all such antigens, this may help explain the specificity of one antibody system versus another in the pathogenesis of disease, in particular tissue damage in the developing fetal heart. LETTERS 2081 Figure 1. Cellular topology of SSA/Ro–SSB/La and lamin B1 in permeabilized and nonpermeabilized apoptotic cultured human fetal cardiomyocytes. For all fields shown, cultured human fetal cardiomyocytes were incubated with staurosporine to induce apoptosis (round condensed nuclei; Hoechst stained [blue]). A–C, Cells were permeabilized prior to staining. A, Apoptotic blebs emerging from the surface were stained by a combination of affinity-purified human antibodies to 48-kd SSB/La, 52-kd SSA/Ro, or 60-kd SSA/Ro (red fluorescence). B, Similar results were observed in permeabilized apoptotic cells stained with anti–lamin B1 antibodies (green fluorescence). In this same field, anti–lamin B1 antibodies stained only the nucleus in the nonapoptotic cell. C, Neither the 3 apoptotic cells nor the single nonapoptotic cell stained with either normal human serum or mouse isotype control. D, Apoptotic cells were not permeabilized prior to staining. Strong red fluorescence indicates surface staining by the affinity-purified anti-SSA/Ro–anti-SSB/La antibodies. In contrast, there is no staining by the anti–lamin B1 antibody. Hoechst crosses intact membranes, accounting for the blue staining in these nonpermeabilized cells. In parallel with the studies presented by Dieudé et al (1), we have confirmed and extended the observation that lamin B1 is redistributed during apoptosis but, unlike SSA/Ro or SSB/La, is not bound by cognate antibodies. Induction of apoptosis included not only staurosporine, the protein kinase C inhibitor used by Dieudé et al, but also the redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). A third method of inducing apoptosis exploited culturing of the cells on poly-(2-hydroxyethyl methacrylate) (poly-HEMA), which results in apoptosis because cells are unable to adhere due to the loss of anchoring signals (3). The target cells were human cardiomyocytes, isolated and cultured from fetuses of gestational age 16–24 weeks. Cells were separately incubated with 0.5 M staurosporine for 7 hours, 0.2 mM DMNQ for 7 hours, or plated on poly-HEMA for 12 hours. Cells were then incubated in the absence or presence of mouse anti–lamin B1 antibody (Chemicon, Temecula, CA) and a combination of affinity-purified human antibodies to 48-kd SSB/La, 52-kd SSA/Ro, and 60-kd SSA/Ro for 30 minutes at 37°C. The media were removed, and the cells were washed twice in phosphate buffered saline with calcium. Cells were fixed with 4% paraformaldehyde for 20 minutes at 22°C. In a second set of experiments the apoptotic cells were first permeabilized with 100% acetone for 60 minutes at 22°C prior to addition of the primary antibodies. The permeabilized and nonpermeabilized cells were subsequently incubated with fluorescein isothiocyanate–conjugated rabbit anti-mouse IgG (for localization of lamin B1) or CY3-conjugated goat anti-human IgG (for localization of SSA/Ro and SSB/La) for 30 minutes at 22°C. Matched isotype controls were negative. In permeabilized nonapoptotic cardiomyocytes, lamin B1 and SSA/Ro–SSB/La antigens were localized to the nucleus 2082 LETTERS with minor cytoplasmic staining, consistent with previous reports (1,4,5, and data not shown). With regard to the latter antigens, it is acknowledged that the topology observed cannot be ascribed uniquely to 52-kd or 60-kd SSA/Ro or to 48-kd SSB/La, given the affinity-purified antisera used for detection. After induction of apoptosis by all 3 methods, lamin B1 as well as SSA/Ro–SSB/La translocated to apoptotic blebs (Figure 1A and B). However, lamin B1, in contrast to SSA/Ro–SSB/La, was not detected at the cell surface (Figure 1D). Taken together, our observations and those of Dieudé et al support discordance in the final cellular destination of translocated nuclear autoantigens during the process of apoptosis. In the case of lamin B1, physiologic noninflammatory clearance of apoptotic cells should proceed uneventfully even in the presence of circulating cognate antibodies. However, in congenital heart block, the maternal anti-SSA/Ro–anti-SSB/La antibodies result in opsonization and inflammatory/fibrotic sequelae. Even if it turns out that SSA/Ro–SSB/La are not unique in this regard, there may be other factors, such as complement binding of certain antigens or degradation of antigens, that facilitate clearing without further sequelae. Establishing the fact that at least one other nuclear autoantigen is not surface-bound during apoptosis of human fetal cardiomyocytes is a step forward. Supported by NIH grant AR-42455. Ms. Chandrashekhar’s work was supported by the Arthritis Foundation, New York chapter. Robert M. Clancy, PhD Hospital for Joint Diseases and New York University School of Medicine New York, NY Edward K. L. Chan, PhD University of Florida Gainesville, FL Sarayu Chandrashekhar, BS Jill P. Buyon, MD Hospital for Joint Diseases and New York University School of Medicine New York, NY 1. Dieudé M, Senécal JL, Rauch J, Hanly JG, Fortin P, Brassard N, et al. Association of autoantibodies to nuclear lamin B1 with thromboprotection in systemic lupus erythematosus: lack of evidence for a direct role of lamin B1 in apoptotic blebs. Arthritis Rheum 2002;46:2695–707. 2. Miranda-Carús ME, Dinu Askanase A, Clancy RM, Di Donato F, Chou TM, Libera MR, et al. Anti-SSA/Ro and anti-SSB/La autoantibodies bind the surface of apoptotic fetal cardiocytes and promote secretion of TNF-␣ by macrophages. J Immunol 2000;165: 5345–51. 3. Clancy RM, Askanase AD, Kapur RP, Chiopelas E, Azar N, Miranda-Carus ME, et al. Transdifferentiation of cardiac fibroblasts, a fetal factor in anti-SSA/Ro-SSB/La antibody-mediated congenital heart block. J Immunol 2002;169:2156–63. 4. Chan EKL, Andrade LEC. Antinuclear antibodies in Sjögren’s syndrome. Rheum Dis Clin North Am 1992;18:551–70. 5. Veldhoven CH, Pruijn GJ, Meilof JF, Thijssen JP, van der Kemp AW, van Venrooij WJ, et al. Characterization of murine-monoclonal antibodies against 60 kD Ro/SS-A and 48 kD La/SS-B autoantigens. Clin Exp Immunol 1995;101:45–54. DOI 10.1002/art.11176 Reply To the Editor: We thank Clancy et al for their interest in our work and for providing us with an opportunity to speculate regarding why some autoantigens are confined within apoptotic blebs while others are expressed at the bleb surface. In a large, multicenter study, we demonstrated the association between the presence of autoantibodies to nuclear lamin B1 in patients with systemic lupus erythematosus (SLE) and protection against thrombosis (thromboprotection). We then aimed to elucidate the mechanism by which autoantibodies to nuclear lamin B1 cause thromboprotection in vivo. Because a number of autoantigens in SLE have been localized specifically to the external surface of apoptotic blebs (1–3), we hypothesized that circulating autoantibodies to nuclear lamin B1 may block the procoagulant effect of apoptotic blebs by binding to lamin B1 displayed at the external bleb surface. Therefore, with biochemical and morphologic studies, we determined the localization of lamin B1 in apoptotic cells and blebs. We used Jurkat cells, a well-studied cell type model for apoptosis, and human umbilical vein endothelial cells. Apoptosis was induced using 2 agents: staurosporine (to induce the mitochondrial apoptotic pathway) and anti-Fas antibody (to trigger the cell death receptor apoptosis pathway). In all cases, lamin B1 was shown to be translocated into surface blebs during apoptosis but was entirely enclosed within the apoptotic bleb membrane. Clancy et al, with microscopy studies, now extend our results to another cell type, cardiomyocytes, which were induced into apoptosis using DMNQ and poly-HEMA. In the figure provided by Clancy et al, corresponding phase-contrast images and scale bars would have been helpful to better identify morphologic structures, especially for the abnormal nuclear morphology observed in Figure 1D, which appears quite different from the nuclear morphology observed in Figures 1A–C. Nevertheless, Clancy et al confirm our results by showing that during apoptosis lamin B1 is relocalized to the blebs but is not recognized by anti–lamin B1 antibodies under nonpermeabilized conditions. Our finding that lamin B1 is not expressed at the surface of blebs is in striking contrast with the finding that other autoantigens, such as Ro and La, are expressed at the surface of blebs. As discussed by Clancy et al, the demonstration of antibody binding to the surface of apoptotic cells is a potentially critical link to the physiopathologic mechanisms applicable to anti-Ro and anti-La, but obviously this is not generalizable to all autoantigens such as lamin B1. These data raise the important question of the predictability of autoantigen expression at the surface versus the interior of apoptotic blebs (i.e., why are certain autoantigens expressed at the surface while others are not?). Autoantigens such as Ro and actin (4) that are translocated at the external surface of the blebs are cytoplasmic. Even the La nuclear antigen is known to shuttle between nucleus and cytoplasm, playing a role in the biogenesis of RNA polymerase III transcripts and translation (5). Indeed, La is predominantly, but not exclusively, immunolocalized to the nucleus in nonapoptotic cells (6). Furthermore, the signal for La to reenter the nucleus, located in the La C-terminus, is cleaved during early apoptosis, causing La accumulation in the cytoplasm (7).