Lupus erythematosus (LE) is a chronic multisystem disease for which a number of immunoregulatory abnormalities have been described. The cause of LE remains unknown, but it is believed to be multifactorial, involving genetic and environmental factors. The disease may vary in severity from limited cutaneous lesions to severe systemic disease; however, in all forms of LE, the skin is one of the main target organs.
In cutaneous LE (CLE), epidermal keratinocytes are considered to be target cells of immunologic injury. A high level of apoptotic keratinocytes in lesional skin from CLE patients has been reported (1, 2). In lupus conditions, the noninflammatory clearance of apoptotic cells seems to be impaired (3, 4), and it has been proposed that the increased rate of apoptosis increases the chance of leakage of intracellular antigens that may trigger an autoimmune response. Furthermore, exposure to ultraviolet B (UVB) irradiation and mediators of inflammation, such as tumor necrosis factor α (TNFα), stimulate keratinocytes to translocate cytoplasmic and nuclear antigens, such as Ro/SSA, to the plasma membrane (5–7).
Aside from their role as target cells in immunologic injury, evidence is accumulating that keratinocytes may play an important role in actively regulating and maintaining the pathologic changes in CLE. Major findings regarding pathologic changes in skin include the expression of Fas (CD95), CD54, and class II major histocompatibility complex (MHC) on keratinocytes in lesional skin (1, 8) and the increased epidermal expression of proinflammatory cytokines, such as TNFα, type I interferons, and high mobility group box chromosomal protein 1 (HMGB-1) (9, 10). However, the hierarchical order of proinflammatory mediators reported to be expressed in the lesional skin of CLE patients remains to be determined.
So far, the “intrinsic” role of human tissue-resident cells such as keratinocytes in the maintenance of chronic inflammation has not been deciphered. In this study, we were interested in determining the potential differences in the proinflammatory response of patient-derived keratinocytes. These cells may be fundamental in influencing the micromilieu and leukocyte activity (11–13) in the skin as the target organ for chronic autoimmune inflammation.
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Tissue-resident cells, such as synovial fibroblasts (23), endothelial cells, and epithelial cells, are being increasingly recognized as active cells in the pathophysiology of various autoimmune diseases. The results presented in this study add new aspects to the notion that keratinocytes are involved in the inflammatory process of cutaneous lesions in patients with LE.
We showed that keratinocytes from patients with CLE display an intrinsically different pattern of immunologic response to IL-18. We found a higher level of apoptosis induction by IL-18 in keratinocytes from CLE patients, but not from normal controls, and we found that this was mediated by high levels of TNFα production and diminished levels of IL-12 release. Thus, IL-18 seems to regulate TNFα as well as IL-12p40. These data support the in vivo data derived from studies of the murine system (24, 25) and indicate that IL-18 may occupy an important position in the proinflammatory cytokine cascade in lupus conditions.
IL-18 is a proinflammatory cytokine (26), and it has also been demonstrated to mediate immune cell infiltration into tissues (27). We have previously shown that primary human keratinocytes respond directly to IL-18, with a prominent effect on the production of CXCL10 (19).
Our results comparing the expression of IL-18 protein in lesional skin from CLE patients with that in skin from healthy controls, as well as the expression of IL-18 mRNA in lesional skin from CLE patients with that in lesional skin from eczema patients, another inflammatory skin disease, indicate high levels of IL-18 expression in CLE skin lesions. A number of studies have found hints of a pathogenetic role of IL-18 in systemic lupus erythematosus (SLE) patients or in murine models of lupus. Increased serum levels have been reported in LE patients (28–30), and high serum levels of IL-18 seem to be correlated with disease activity (31, 32). Amerio et al (32) proposed that the role of IL-18 in the pathogenesis of SLE might be important through its apoptosis-mediating properties. An up-regulation of IL-18, which was also expressed by tubular epithelial cells, has been reported (33) and recognized as a hallmark of lupus nephritis (34). Interestingly, MRL/lpr mice showed clear benefit with regard to glomerulonephritis, renal damage, and mortality rates from the targeting of IL-18 with cDNA vaccination (24), whereas administration of IL-18 to MRL/lpr mice resulted in accelerated proteinuria, glomerulonephritis, vasculitis, raised levels of proinflammatory cytokines, and the development of a butterfly facial rash (25). Thus, there may be important parallels in the pathogenesis of skin and renal inflammation. In both conditions, IL-18 may play a prominent role in the cytokine hierarchy in the local inflammatory micromilieu at epithelial “interfaces.” However, more data are clearly needed to prove this hypothesis.
The most prominent effect of IL-18 on keratinocytes from CLE patients that was identified in this study was the increased production of TNFα. From our data, we cannot conclude whether the increased TNFα production in CLE keratinocytes is due to increased sensitivity to IL-18 (and thus dysregulation at the level of IL-18 “signaling”) or altered regulation of TNFα (e.g., at the promoter level). An IL-18–mediated increase in TNFα has been described in relation to synovial macrophages (35), human peripheral blood mononuclear cells (36), and in murine trinitrobenzene sulfonic acid–induced colitis (37). Sullivan et al (38) evaluated chromatin at the TNFα locus within the monocyte population of SLE patients as compared with healthy controls and found more highly acetylated histones at the TNFα locus in monocytes from SLE patients. An increased transcriptional competence of TNFα could also play a role in IL-18–stimulated CLE keratinocytes.
However, we did not observe a correlation between increased TNFα secretion and the known –308 A polymorphism in the TNFα promoter. It has been reported that LE (39), subacute cutaneous LE (17), as well as other autoimmune diseases are associated with that polymorphism. This SNP has been associated with enhanced TNFα production, particularly after UVB irradiation (17). However, the issue remains a subject of controversy, since other groups of investigators, such as Popovic et al (9), did not find higher expression of TNFα in either affected or unaffected skin from CLE patients carrying the A allele as compared with patients carrying the GG genotype. It is clear that many patients with LE do not carry the –308 A polymorphism in the TNFα promoter, indicating the involvement of other regulatory factors. Based on recently published studies (20, 40), we also analyzed the IRF5 rs2004640 genotype, but again, we found no association between the IRF5 rs2004640 T allele and high levels of TNFα production. Of note, with regard to the functional significance of IRF5, Kozyrev et al (40) suggested that there may be other functional polymorphisms in IRF5 that are yet to be identified. Probably an interaction between different SNPs may be required to lead to functionally relevant alterations in immune responses.
Our results showed a high level of TNFα expression along with a low level of IL-12 expression upon stimulation of CLE keratinocytes with IL-18. As for the role of IL-12 in lupus conditions, there are some conflicting data. However, a number of studies showed reduced production of IL-12 by blood-derived cells from patients with SLE (28, 41–45). So far, no studies analyzing the expression of IL-12 in the skin of patients with LE inflammation have been published. Importantly, Werth et al (46) demonstrated that IL-12 completely blocks UV irradiation–induced secretion of TNFα from skin keratinocytes and fibroblasts. Thus, the low expression of IL-12 observed in our studies could contribute to the increased TNFα production seen in response to IL-18 in CLE keratinocytes. IL-12 was previously found to inhibit gene transcription and IL-10 release from UVB-irradiated keratinocytes (47) as well as UVB irradiation–induced apoptosis and DNA damage (22). Werth et al (46) suggested that IL-12 might play an important role in healthy individuals by decreasing TNFα-mediated apoptosis of keratinocytes, thereby diminishing one source of self antigen.
Dysregulation of programmed cell death and impaired removal of apoptotic cells have been discussed as pathogenetic factors in LE. According to the “clearance hypothesis,” the number of apoptotic cells in LE target organs may overwhelm the body's capacity to clear them. The role of IL-18 in apoptosis is a subject of controversy in the literature. While Schwarz et al (48) found IL-18 to be a protective cytokine, other investigators found IL-18 to be a proapoptotic mediator (49) in human endothelial cells. Like many other effects of IL-18, the predominant outcome may depend on the cell type, the species examined, and the surrounding milieu.
Apart from IL-18, other cytokines, such as CXCL10, type I and type II IFNs, HMGB-1, and TNFα (9, 13), have been described as effector molecules in CLE. Our data support the idea that IL-18, acting as a proximal regulatory cytokine, may control the micromilieu response pattern in the skin compartment. Therefore, the findings of this study support the notion that targeting the bioactivity of IL-18 may be a promising immunopharmacologic intervention in the treatment of LE conditions (involving the kidney or skin inflammation) (50). IL-18 binding protein (51) is one of the compounds that might prove clinically useful in LE therapy.
In conclusion, our findings suggest that IL-18 may play an important role in triggering inflammation in CLE, promoting a cytokine imbalance toward a high TNFα response and a low IL-12 response, thus providing a proapoptotic microenvironment for keratinocytes. Since the keratinocytes used in our study were not derived from lesional skin, the different response pattern to IL-18 must be based upon intrinsic differences in CLE keratinocytes as compared with healthy keratinocytes.