Published online before print September 2, 2003
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Hospital for Joint Diseases, New York University School of Medicine, Department of Rheumatology, New York
1 Correspondence: Hospital for Joint Diseases, New York University School of Medicine, Department of Rheumatology, 301 E. 17th Street, Room 1600, New York, NY 10003. E-mail: bobdclancy{at}aol.com
Key Words: SSA/Ro antibodies • congenital heart block • tumor necrosis factor-
Abstract: Transforming growth factor-ß (TGF-ß) has been considered an anti-inflammatory cytokine responsible for the bland removal of apoptotic cells. What is less established is the extent of secretion of this cytokine during the clearance of opsonized apoptotic cells via Fc
-mediated uptake. To date both decreased (favoring predominance of inflammation) and increased (favoring resolution of inflammation but potentially pro-fibrotic) responses have been demonstrated. In an in vitro model of autoantibody-induced cardiac injury, we herein demonstrate that macrophages cocultured with apoptotic human fetal cardiocytes bound by anti-SSA/Ro antibodies secrete increased levels of TGF-ß. Prolonged secretion of this cytokine may contribute to the exuberant scarring seen in congenital heart block associated with maternal autoantibodies reactive with SSA/Ro and SSB/La antigens.
As Brown et al. [1 ] suggest, "Quantitative and kinetic balance of pro- and anti-inflammatory mediators produced locally determines the safe outcome of many tissue processes and contributes to immunological homeostasis". Notable in Figure 4D of Brown et al. [1 ], TGF-ß secretion by healthy donor macrophages increased from undetectable levels to approximately 150 pg/ml when incubated with nonopsonized apoptotic neutrophils but increased to approximately 600 pg/ml when incubated with opsonized apoptotic neutrophils. Although the significance of this finding might not be immediately intuited, it is notable that excessive and prolonged production of TGF-ß can result in tissue fibrosis [4 ]. Although rarely acknowledged, sustained secretion of TGF-ß could well be injurious with regard to scar potential. The long-term effects of TGF-ß production and/or other growth factors induced by altered processing of apoptotic cells might ultimately affect wound healing and fibrosis.
Our laboratory has approached the study of inflammatory-fibrotic processes from a totally different perspective than that of Brown and her coworkers [1 ]. Instead of addressing the consequence of diminished production of TGF-ß, as was observed in patients with CGD, we have studied the role of excessive TGF-ß as a profibrotic mediator in a model of passively acquired autoimmunity: SSA/Ro-associated congenital heart block [5 ]. This fetal/neonatal autoimmune disease in pregnancy is characterized by fibrotic replacement of the atrioventricular node. The maternally derived anti-SSA/Ro antibodies are transported across the placenta and are necessary but not sufficient to cause disease [6 ]. One of the proposed hypotheses to explain the pathogenicity of maternal anti-SSA/Ro antibodies is that apoptosis of fetal cardiac-conducting cells and/or working myocytes results in translocation of intracellular SSA/Ro to the cell surface where it can be bound by cognate autoantibodies. These apoptotic cells become opsonized analogous to the UV-exposed neutrophils rendered opsonized by labeling with anti-human CD45, as in the experiments of Brown and colleagues [1 ]. The question addressed in our study was the role of TGF-ß in promoting scar formation. Indeed, it was demonstrated that TGF-ß induced the expression of smooth muscle actin (SMAc), a marker for transdifferentiation of cultured human fetal cardiac fibroblasts to myofibroblasts, a scarring phenotype [5 ]. Supernatants from human macrophages cocultured with human fetal cardiocytes, rendered apoptotic by incubation on poly-hema-coated plates [5 ] and opsonized by incubation with affinity-purified anti-52 and -60 kD SSA/Ro, markedly up-regulated the expression of SMAc on separately cultured cardiac fibroblasts. Anti-TGF-ß-neutralizing antibodies inhibited this increased expression, supporting that TGF-ß is present in the supernatants and is activated. This up-regulation was not observed using supernatants from macrophages cocultured with apoptotic cardiocytes incubated with IgG from a healthy donor (nonopsonized). Furthermore, as shown in Figure 1 (previously unpublished), the mean TGF-ß secreted from human macrophages cocultured with the opsonized apoptotic cardiocytes, nonopsonized apoptotic cardiocytes, or no coculture was 651 pg/ml, 319 pg/ml, and 71 pg/ml, respectively (P=0.03; opsonized apoptotic cardiocytes vs. macrophages alone). In these experiments, the in vivo state of activation/latency of the total TGF-ß was not determined, as the supernatants were acid-activated prior to testing.
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Figure 1. Cytokine release from human macrophages cocultured with nonopsonized and opsonized apoptotic human fetal cardiocytes for 24 h. Macrophages (M ) were incubated alone (solid bars) with poly-hema-treated apoptotic cardiocytes, nonopsonized (hatched bars), and opsonized (open bars) with human affinity-purified anti-52 and -60 kD SSA/Ro antibodies and cytokines [tumor necrosis factor (TNF-), upper; TGF-ß, lower], measured from supernatants. Data are displayed as mean + SEM. P = 0.03, macrophages alone versus cocultured with opsonized apoptotic cardiocytes for secretion of TGF-ß.
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to counter-balance the inflammatory response in some circumstances. Accordingly, we envisage that under "physiologic" conditions, the clearance of dying cells is noninflammatory, consistent with current dogma, and that the secretion of TGF-ß and/or its activation is ephemeral. In diseases such as CGD, dysfunctional clearance of apoptotic cells associated with diminished production of TGF-ß may tip the balance in favor of inflammation. In contrast, increased TGF-ß secretion may over-ride an anti-inflammatory effect and tip the balance in favor of fibrosis. As most inflammatory lesions do not progress to fibrosis, one would predict that in autoimmune-mediated congenital heart block, there is inadvertent opsonization of the apoptotic cardiocyte, resulting also in dysregulation of cell clearance, but unlike CGD, the dysfunction is characterized by excessive release of active TGF-ß (or activated by another cell). The net effect undermines the normal healing process, resulting in irreversible scar. Accordingly, ingestion of apoptotic cells and subsequent secretion of TGF-ß by well-intended macrophages may result in opposing consequences in health and disease, depending in part on the presentation of the "meal" and the "utensil" used for eating.
ACKNOWLEDGEMENTS
This work was supported in part by National Institutes of Health Grant Nos. AR42455 and AR48409.
Received June 16, 2003; revised July 11, 2003; accepted July 16, 2003.
REFERENCES
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