Nearly a quarter of a century ago, Franco and colleagues reported that maternal anti-SSA/Ro antibodies were associated with a neonatal skin rash that resembled subacute cutaneous lupus erythematosus (SCLE) (1), an immunologic finding that would later prove important in relation to congenital heart block (CHB) as well. It is now established that rash and CHB, despite the former being transient and the latter permanent, are each major manifestations of the so-called neonatal lupus syndromes. The disparate timing and clinical implications of neonatal lupus rash and CHB suggest that both fetal and environmental factors contribute to the specific tissue injury evoked by the maternal autoantibodies. In cutaneous disease, a major clue to elucidation of the pathogenesis may be the photoinductive nature of the rash. The notion that photosensitivity has an influence is supported by the findings of 2 major studies, one of which showed that in 45 of 48 cases (94%), rashes appeared after or were exacerbated by exposure to ultraviolet (UV) light (2). In the other study, photosensitivity was a prominent feature, observed in 12 of 18 children (67%) (3).
UVB light (290–320 nm) is the exogenous trigger that is most often responsible for the skin lesions of cutaneous LE (4). There are several pathologic mechanisms that may be relevant to the induction and/or potentiation of these lesions in the setting of autoimmunity. UV light promotes tumor necrosis factor α (TNFα)–induced apoptosis in cultured keratinocytes. Although this has not been formally demonstrated in keratinocytes, apoptosis in cardiocytes redistributes nuclear antigens to the surface membrane, which can result in binding of nonpermeabilized cells by cognate extracellular antibodies, e.g., anti-SSA/Ro and SSB/La. Macrophages that phagocytose these opsonized apoptotic cells can release TNFα, favoring an inflammatory response (5). Of relevance, UVB can also directly trigger release of TNFα from keratinocytes and dermal fibroblasts (6).
There is variability in the UV-induced TNFα secretion by different transformed keratinocyte lines, suggesting that human polymorphisms may also be contributory. The gene encoding TNFα is highly polymorphic, and a substitution of G to A at position −308 in the promoter region (TNF2) has been associated with increased production of this cytokine. The common (wild-type) allele, −308G (TNF1), has a frequency of ∼80% in Caucasians and 92% in African Americans (7). Werth and colleagues recently demonstrated that treatment with UVB plus interleukin-1α caused a 300-fold increase in chloramphenicol acetyl transferase transcription over baseline with the −308A promoter, compared with <15% over baseline with the −308G promoter (8). However, it is acknowledged that specific cell type and stimuli may be influential in the differential expression of the −308 promoter polymorphisms. Of clinical relevance, an increased prevalence of −308A has been observed in SCLE, an extremely photosensitive form of cutaneous LE, and in adult dermatomyositis (DM) (8), another photosensitive disease, but not in discoid LE (DLE), a less photosensitive form of lupus.
The link between anti-SSA/Ro antibodies, photosensitivity, and TNFα promoter polymorphisms extends to HLA class II molecules. In Caucasians, there is strong linkage disequilibrium between the −308A allele and HLA–DRB1*03 (9). The presence of DRB1*03, as well as DQB1*02, is also common in individuals who are positive for anti-SSA/Ro and SSB/La antibodies (10). Accordingly, it is of note that in the recent studies by Werth and coworkers, the proportion of Caucasians with at least 1 −308A allele who also carried the HLA–DRB1*03 allele was 100% among SCLE patients (who are often anti-SSA/Ro positive) but only 60% among DM patients (who are infrequently anti-SSA/Ro positive) and 56% in the control group (8).
Given these recent insights into molecular and genetic features of 2 photosensitive autoimmune rashes, this investigation was initiated to explore the role of TNFα in the pathogenesis of cutaneous neonatal lupus. Extensive studies were performed to address whether the frequency of the TNFα −308A allele in association with HLA–DQB1*02 and DRB1*03 is increased in children with neonatal rash compared with either children with CHB alone or children who are completely healthy but have been exposed in utero to maternal anti-SSA/Ro antibodies. In addition, immunohistologic studies were performed on available lesional skin from 3 infants born to mothers with anti-SSA/Ro antibodies, in order to establish the protein expression of TNFα.
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- PATIENTS AND METHODS
Since rash occurs in only a minority of children exposed to maternal anti-SSA/Ro and SSB/La antibodies in utero, it is likely that a genetic predisposition is operative. Given the clinical resemblance of SCLE to the neonatal lupus rash, the TNFα –308A promoter polymorphism (associated with high cytokine production) may be one important determinant contributing to the pathogenesis of the cutaneous disease. The following observations support this hypothesis: 1) the majority of children with rash had the TNFα –308A promoter allele; 2) this allele was associated with the presence of both HLA–DQB1*02 and HLA–DRB1*03 in children with rash significantly more often than in anti-SSA/Ro–exposed children without rash; and 3) TNFα was demonstrated in lesional skin.
The proposed pathologic cascade to rash may be initiated by UVB light or another unknown exogenous stimulus that results in increased dermal fibroblast and keratinocyte secretion of TNFα. Cells from neonates with the –308A allele may have increased secretion of this inflammatory cytokine (perhaps even obviating the need for photoinduction), given the preferential binding of specific transcription factors to this promoter polymorphism. The secreted TNFα may augment keratinocyte (6) and dermal fibroblast apoptosis. In turn, apoptotic cells are a source of intracellular antigens, such as SSA/Ro and SSB/La, that may be bound by circulating maternal autoantibodies, thus rendering the cells “opsonized.” Keratinocytes express CD16 (Fcγ receptor III [FcγRIII]), and FcγR-mediated uptake of opsonized apoptotic cells represents an important pathologic pathway involving clearance of opsonized apoptotic cells, in addition to contributing to the synthesis/release of TNFα by FcγR-dependent signaling. For example, Miranda-Carus et al demonstrated that macrophages release TNFα during the clearance of opsonized apoptotic cardiocytes (5). Accordingly, the extensive staining for TNFα may be due to both UVB induction and secretion by inflammatory cells.
It is tempting to speculate that the association of –308A, DQB1*02, and DRB1*03 in children with neonatal lupus rash has functional consequences with regard to pathogenesis. The frequency of these 3 alleles alone and/or together was significantly higher than in a previously reported healthy control group of 93 Caucasians, in which the prevalence of DRB1*03 was only 17% and that of DQB1*02 was 21% (13), as well as in another group of 210 healthy controls (93% Caucasian, 7% African American), in which the prevalence of both DRB1*03 and –308A was 16% (13). It is readily acknowledged that use of a larger sample, association tests such as transmission disequilibrium testing, and ethnically and geographically matched controls for each family would strengthen the genetic findings presented herein. However, these limitations notwithstanding, the most compelling genetic association was that the children with rash had a significantly higher prevalence of the combination of –308A, DQB1*02, and DRB1*03 than did children without rash who were also exposed to maternal anti-SSA/Ro antibodies. It is also of note that the prevalence of the −308A;DQB1*02;DRB1*03 combination was greatest in the neonatal lupus mothers. The high prevalence of DRB1*03 confirms and extends previous findings (13) and is not surprising given the well-established association of anti-Ro/La antibodies with the presence of DQB1*02 and DRB1*03 (10, 14).
Taken together, these findings indicate that the same extended haplotype might contribute to a “double hit,” one in the mother and one in her offspring. The HLA portion of the extended haplotype DQB1*02;DRB1*03 provides the genetic predisposition for the generation of the candidate autoantibodies, which cross the placenta and bind apoptotic neonatal keratinocytes and fetal cardiocytes, thus initiating an inflammatory response. The TNFα portion of the extended haplotype in the children may further contribute to tissue injury by amplifying the inflammatory cascade, a notion supported by the limited immunohistologic results presented. The availability of nonlesional skin from neonates exposed to maternal anti-Ro/La, and demonstration that TNFα was not expressed therein, would further support the pathologic relevance of the TNFα staining in the lesional skin.
In terms of the morphology of the photoeruption and the histology of the skin lesions, the rash of neonatal lupus is remarkably similar to that of SCLE, another photosensitive autoimmune skin disease. In SCLE there is also basal cell damage in the epidermis and a superficial mononuclear cell infiltrate in the upper dermis (15). The notion of a similarity in pathogenesis between the rash of neonatal lupus and SCLE is supported by genetic evidence from the present study and that by Werth et al (8). While a high frequency of –308A has been noted in patients with DM, DRB1*03 was not increased, likely due to the absence of anti-Ro/La antibodies in that disease. Neither the –308A allele nor DRB1*03 was associated with DLE, a less photosensitive rash in which anti-Ro/La antibodies are infrequently observed. Furthermore, the characteristic lesions of DLE, such as follicular plugging, dermal atrophy, and scarring, are not typical of neonatal lupus or SCLE.
In summary, through the use of a translational approach to evaluate genetic factors in the context of histologic features, this study has provided insights into the pathogenesis of the cutaneous manifestation of neonatal lupus,. The finding that the TNFα –308A polymorphism in association with HLA–DQB1*02 and DRB1*03 influences disease expression in the setting of a common exposure to a risk factor, in this case maternal antibodies to Ro/La, is of biologic interest and complements the histologic findings. Although proof of concept must await application in an animal model, our results indicate that TNFα is an important neonatal susceptibility factor, and a component of its synthesis/secretion may be a target in future strategies for treatment.