Novel Insights into the Role of S100A8/A9 in Skin Biology

Abstract

S100A8 and S100A9 belong to the damage associated molecular pattern molecules. They are upregulated in a number of inflammatory skin disorders. Due to their abundance in myeloid cells the main function of S100A8/A9 has been attributed to their role in inflammatory cells. However, it is becoming increasingly clear that they also exert important roles in epithelial cells. In this review we discuss the context-dependent function of S100A8/A9 in epithelial cells and its impact on wound healing, psoriasis and other skin diseases.

Introduction

S100A8 (calgranulin A or migration inhibitory factor-related protein 8; MRP-8) and its binding partner S100A9 (calgranulin B or MRP-14) are members of a multigenic family of non-ubiquitous cytoplasmic Ca²⁺-binding proteins ¹. They received strong interest over the last years due to their characterization as damage associated molecular pattern molecules (DAMPs) ², their differential expression in chronic inflammatory diseases, and their association with cancer ³. S100A8 and S100A9 are expressed and specifically released from activated phagocytes ^4,5, and patients suffering from (auto)immune disorders such as type 1 diabetes ⁶, cystic fibrosis, chronic bronchitis, rheumatoid arthritis, multiple sclerosis, sepsis, autoimmune colitis and psoriasis ⁷ have high S100A8/A9 plasma levels.

S100A8 and S100A9 preferentially form heteromeric protein complexes, termed S100A8/A9 or calprotectin, but distinct functions for the individual S100 proteins have also been reported ^8,9. Constitutive expression of S100A8/A9 is largely restricted to myeloid cells such as neutrophils, monocytes, macrophages, and some other myeloid cells (E-Supplement 1), in contrast to other related cells such as lymphocytes, basophils, and eosinophils, which do not ²². S100A8/A9 comprise about 30% to 60% of all cytosolic proteins in neutrophils and about 1–5% of all monocyte cytosolic protein ²³. During myeloid differentiation the S100A8/A9 expression is tightly regulated ²⁴. Recently, it was demonstrated that in a mouse autoimmune model, S100A8/A9 production was essential for the induction of autoreactive CD8⁺ T cells and the development of systemic autoimmunity ²⁵. Whereas Cd40lg-transgenic mice developed autoimmune dermatitis and overt disease, Cd40lg × S100A9^−/− mice showed a delayed onset and significantly reduced severity of dermatitis. Adoptive cell transfer experiments indicated that S100A8/A9 expression was required for functional development of autoreactive CD8⁺ T cells.

S100A8/A9 is complex in its actions as it has both intracellular and extracellular functions (E-Supplement 2). Its role has previously been shown in the propagation of inflammation and the upregulation of proinflammatory cytokines, and some of the extracellular functions are mediated by toll-like receptor 4 (TLR4) ^30,40 or the receptor for advanced glycation end-products (RAGE) ^37,38,39. However, other studies also reveal anti-inflammatory actions ⁴⁴.

Expression of S100A8 and S100A9 can also be induced in non-myeloid cells, such as skin cells, as well as in tumors of epithelial origin (E-Supplement 3). Together, these observations indicate a role for S100A8 and S100A9 in the pathophysiology of certain skin diseases.

S100A8/A9 play important roles in non-myeloid cells

Although the focus of interest currently lies on the role of S100A8/A9 in inflammatory cells, there is also growing evidence for an important role of these S100 proteins in non-myeloid cells ¹⁸. Whole-body S100a9-deficiency was previously demonstrated to reduce atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice through reduced arterial macrophage accumulation ²⁹. Because of the abundance of S100A8/A9 in myeloid cells compared to non-myeloid cells, myeloid-derived S100A8/A9 was believed to mediate the effects of whole-body S100A9-deficiency. To test this hypothesis, low-density lipoprotein receptor-deficient (Ldlr^−/−) mice were transplanted with bone marrow from S100a9^−/− mice or wild type littermate controls and then fed a high-fat diet for 20 weeks. However, bone marrow S100A9-deficiency did not reduce atherosclerosic lesions or arterial macrophage or neutrophil accumulation ¹⁸. These data could be explained by a more important role of S100A8/A9 in non-myeloid cells than previously recognized. Accordingly, S100A8/A9 are expressed in both vascular smooth muscle cells and endothelial cells, and are believed to exert pro-atherosclerotic effects in these cell types ^37,68,69. It is possible that the effect of S100A8/A9-deficiency on various pathologies and diseases depends on the relative contribution of different cell types at specific stages of disease progression. In view of these novel findings, the role of S100A8/A9 expressed in epithelial cells in wound healing (and other skin diseases) needs reevaluation.

S100A8 and S100A9 expression in psoriasis

A number of studies support the important role of S100A8/A9 in psoriasis. S100A8 and S100A9 were found in lesional skin of psoriasis patients but expression was low in nonlesional skin from the same individual from which lesion sample was obtained ⁷⁰. Consistently, S100A8/A9 serum levels are elevated in psoriatic patients, and elevated S100A8/A9 serum levels track disease activity, suggesting that these S100 proteins are potential mediators in psoriasis ⁷¹. Linkage analyses identified a disease susceptibility region, the PSORS4 locus, which is mapped to chromosome 1q21 ⁷². Close to this region, the S100 gene cluster is located harboring both S100a8 and S100a9 genes together with several epidermal differentiation markers, such as profilaggrin and involucrin ^73,74.

In two transgenic mouse model of psoriasis, S100a8 and S100a9 gene expression was shown to be an early molecular event in the development of psoriasis, and induction of S100a8/a9 gene expression occurred well before any histological alterations or deregulation of cytokines was observed. These models reveal evidence that either Jun proteins (JunB and c-Jun) or STAT3 are involved in control of S100A8/A9 expression during skin inflammation ^75,76.

High levels of either IL-22 ⁴⁹ or epithelium-derived growth factor ⁷⁷ in psoriatic skin might also contribute to the increased expression of S100A8/A9. In addition, oncostatin M (OM) is found in psoriatic skin and suppresses the expression of the “classical” epidermal differentiation markers, but induces S100A8/A9 ⁷⁸. S100a9 promoter analysis provided strong evidence that OM induces S100A9 through the STAT3-signaling cascade, although direct binding of STAT3 to the promoter again was not observed, suggesting that STAT3 might regulate S100A9 induction through an indirect mechanism ⁷⁹. Further research is needed to elucidate the exact mechanism of the upregulation, and more importantly, whether S100A8/A9 play a causative role in psoriasis.

Is S100A8/A9 involved in the keratinocyte response to injury?

Wound healing represents the outcome of a large number of interrelated biological events that are orchestrated over a temporal sequence in response to injury and its microenvironment (Fig. 1). Resident cells (i. e. keratinocytes and fibroblasts) and infiltrating leukocytes participate differentially in the three well-defined phases of wound healing: the inflammatory phase, the proliferative phase, and the remodeling phase. These interrelated biological events are also characterized by a spatial and temporally expression pattern of genes ⁸⁰.

Figure 1.

Schematic representation of cell types involved in wound healing and the potential effects of S100A8/A9 on those cells. In neutrophils, S100A8/A9 promote expression of inflammatory mediators, phagocytosis, oxidative burst and migration through the epithelium. Likewise, in monocytes, S100A8/A9 stimulate migration and an inflammatory phenotype. Macrophages express and release significantly less S100A8/A9 than do monocytes. Keratinocytes express S100A8/A9 following exposure to inflammatory stimuli, and S100A8/A9 expression results to decreased proliferation, enhanced ROS generation, differentiation and migration as well as increased expression of proinflammatory cytokines and matrixmetalloproteinase-9. When added exogenously, S100A8/A9 promote proliferation of keratinocytes and fibroblasts, which might contribute to the proliferative phase. Thus, S100A8/A9 affect the major cell types involved in wound healing, and these proteins have cell type-selective effects.

Neutrophils and monocytes play important roles in all stages of tissue repair. These cells are rapidly recruited to the injury to perform several host-defense functions (Fig. 1). Macrophages also produce numerous cytokines, growth and angiogenic factors that are believed to play important roles in the regulation of bro-proliferation and angiogenesis (Fig. 1). However, data from various knockout and knockdown studies in mice demonstrate that inflammatory cells are not absolutely essential for efficient wound healing ⁸¹. Consistently, S100A9-deficient mice display an approximate 4-fold reduction of infiltrating granulocytes in 5-day wounds but accelerated wound closure ⁹. It is thus possible that expression of S100A8/A9 in epithelial cells act to slow wound healing, for example by reducing keratinocyte proliferation and inducing differentiation, as discussed below.

S100A8/A9 are found in differentiating suprabasal wound keratinocytes ⁴⁶, especially in the first 12 to 24 hours after injury, with a gradual return to baseline expression over a 2-week period ⁸². Thus, during wound healing, there is a rapid increase of S100A8/A9 immunoreactivity that maps infiltration of recruited leukocytes (primarily neutrophils) followed by a sustained S100A8/A9 expression in wound keratinocytes that represent de novo-synthesis of S100A8/A9 (Fig. 1). An interesting study on wild-type versus Pu.1^−/− mice demonstrates that both keratinocytes and leukocytes express S100A9 (and S100A8) in wounds of wild-type mice, whereas S100A9 expression is limited to keratinocytes in wounds of Pu.1^−/− mice ⁸³. The macrophage-restricted transcription factor Pu.1 is required for normal adult myelopoiesis. Pu.1 gene disruption affects the maturation of progenitors of the neutrophil or monocytic lineage and, therefore, the induction of S100a9 gene expression during hematopoietic development ^84,85. In wounds of wild-type mice, the S100A8/A9 proteins were expressed as early as 3 hours after injury, with expression peaking at 12 hours post-wounding, whereas in wounds from Pu.1^−/− mice, expression was not observed until 12 hours post-wounding and S100A8/A9 levels were much reduced compared with that of wild-types. In situ hybridization clearly showed S100A8 and S100A9 to be expressed, in addition to keratinocytes, in the region of the wound populated by inflammatory cells in wild-type mice only. Interestingly, wounds in neonatal Pu.1^−/− mice, which are essentially missing both neutrophils and macrophages, show both speedier repair and reduced scarring, consistent with the results from S100A9-deficient mice ⁹. Furthermore, S100A9 is increased in nonhealing as compared with healing wounds. In the healing wound S100A9 staining is primarily observed in suprabasal keratinocytes at the hyperproliferative wound edge. In nonhealing wounds, on the other hand, S100A9 is observed in the papillary dermis, suprabasal layers of the hyperproliferative wound edge, and to some extent in CD11b positive myeloid cells ⁸⁶.

Together, these studies suggest that S100A8/A9 expression in both inflammatory cells and epithelial cells is likely to play an important role in wound healing. Bone marrow transplant studies, using S100A9-deficient mice as donors would shed light onto the relative contribution of S100A8/A9 expression in inflammatory cells versus epithelial cells in wound healing.

What biological functions can be attributed to epithelial S100A8/A9?

Over-expression studies demonstrated that intracellular S100A8/A9 promote epithelial NADPH oxidases and subsequently NF-κB activation, consistent with the view that NF-κB is a redox-sensitive transcription factor ⁸⁷. The NF-κB pathway has important roles in the regulation of keratinocyte growth, survival and differentiation. In contrast to immune cells, NFκB activation induces growth arrest in epithelial cells ⁸⁸. Consistently, S100A8/A9 over expression impairs cell proliferation, survival and differentiation ⁸⁹ (Fig. 1). Indeed, S100A9 expression in keratinocytes correlates with the degree of differentiation ⁹⁰. Increased differentiation and reduced proliferation of keratinocytes in response to S100A8/A9 might of importance for those cellular processes, including ultraviolet (UV) irradiation, virus infection and wound healing, in which transiently expression of S100A8/A9 is induced.

UV-induced skin carcinogenesis is partly prevented by overexpression of S100A8/A9. Similar, growth arrest and apoptosis are part of the innate antiviral immune response. In these conditions, the S100A8/A9-suppressed cell growth might represent an additional checkpoint for cells either to set about repairing themselves or to commit suicide through apoptosis. Upon skin injury, the proliferation and migration of keratinocytes is required for wound re-epithelialization and healing. During wound healing, keratinocytes at the border of the wound recapitulate part of the epithelial-mesenchymal transition (EMT) process. They appear to acquire an intermediate phenotype known as the “metastable” state, which allows them to move while maintaining loose contacts rather than migrating as individual cells ⁹¹. There are several findings that indicate a stimulatory role for S100A8 and S100A9 in epithelial migration (Fig. 1). First, S100A8/A9 are expressed in wound keratinocytes ⁴⁶. Second, the genes encoding the S100 family are found to be localized in a cluster on human chromosome 1q21 close to the human epidermal differentiation complex ^73,74, and it has been suggested that expression of these genes could be coordinately regulated ⁹². Third, conversion to a migratory keratinocyte phenotype coincides with MMP-9 induction ^93,94, and MMP-9 gene expression is significantly enhanced in S100A8/A9-over expressing HaCaT keratinocytes (E-Supplement 4). Fourth, S100A8/A9 expression protects from anoikis ⁸⁹. Fifth, S100A8/A9 bind to keratin filaments ⁹⁵, and there is evidence for their association with the epidermal cytoskeleton in inflammatory dermatoses ⁹⁶.

Importantly, EMT features in cutaneous wound healing differ from the features of cancer metastasis because epithelial cells involved in wound healing that acquire mobility and mesenchymal phenotypes return to the epithelial phenotype, whereas epithelial cells involved in cancer metastasis do not. However, the enhanced Akt phosphorylation in S100A8/A9-positive HaCaT keratinocytes ⁸⁹ might represent a link between wound healing and the development of human Squamous Cell Carcinoma (SCC). Akt activation may cause apoptotic resistance and thus contribute to malignant transformation in response to chronic UV exposure ⁹⁷. In deed, S100A8 and S100A9 are prominent differentially regulated genes in SCC ³.

Together, the findings to date suggest that S100A8/A9 have important functions in epithelial cells, including enhancing keratinocyte differentiation while inhibiting proliferation and anoikis, enhancing keratinocyte migration, in part by stimulating MMP-9 expression. Further studies are required to elucidate whether the effects of S100A8/A9 are mediated by intracellular or extracellular mechanisms in keratinocytes. S100A8/A9 have been shown to be secreted by epithelial cells ⁴⁶, and extracellular S100A8/A9 exert mitogenic activity on keratinocytes, probably through activation of TLR4 and/or RAGE ⁹⁸. Of note, S100A8/A9 release from intact non-myeloid cells is significantly lower than that from myeloid cells, suggesting that the biological effects of S100A8/A9 expressed by epithelial cells might be largely intracellular, unless membrane integrity is compromised.

Correlation of epithelial S100A8/A9 expression and skin diseases

S100A8/A9 are abundant in psoriasis keratinocytes ⁷⁰, which demonstrate abnormal differentiation and hyperproliferation, yet in vitro S100A8/A9 overexpression studies have shown that S100A8/A9 mediates keratinocyte differentiation and inhibit proliferation ⁸⁷. How can these seemingly contradictory findings be explained? It is possible that the increased expression of S100A8/A9 in psoriasis keratinocytes provide a negative feedback on the hyperproliferative state of these cells. It is also possible that the cell culture overexpression studies do not accurately mimic the effects of S100A8 and S100A9 in vivo in psoriatic skin. A third possibility is that S100A8/A9 secreted from keratinocytes ⁴⁶ exert mitogenic activity on these cells ⁹⁸ whereas intracellular S100A8/A9 mediate growth inhibition and promote differentiation. In addition, it is well known that the presence of proinflammatory cytokines released from activated keratinocytes and various immune cells stimulate keratinocyte proliferation and amplifies the hyperproliferative response to barrier disruption ^51,52.

In atopic dermatitis (AD), S100A8/A9 expression is below detection levels ²⁵. Pimecrolimus is a calcineurin inhibitor that has successfully been used in the treatment and prevention of recurrence in patients with AD. It has recently been shown that this drug induces the up-regulation of S100A8/A9 and other genes that are essential for the normal barrier function of skin ⁹⁹. Further studies are required to exlore whether S100A8/A9 takes part in mediating the beneficial effect of pimecrolimus.

Conclusion and perspectives

The present review demonstrates the complexity of S100A8/A9 function in skin biology and disease given by the expression patterns of S100A8/A9 in myeloid cells and dermal cells as well as the diverse intracellular and extracellular functions of S100A8 and S100A9. The biological role of S100A8/A9 is predicted to depend on the relative contribution of the different cell types at specific stages of disease progression. S100A8/A9 are expressed in epithelial cells in response to stress. In conditions such as wound healing and UVB irradiation in which S100A8/A9 are transiently induced, their intracellular effects may predominate. In psoriasis and other skin diseases in which the proteins are abundant in dermal cells and the epithelial barrier is compromised, the effects of S100A8/A9 may be mainly mediated through activation of TLR4 and/or RAGE by extracellular S100A8/A9.

Recent studies demonstrate that S100A8/A9 have opposite effects in inflammatory cells. S100A8/A9 promote an inflammatory phenotype in neutrophils, while suppressing an inflammatory phenotype in DCs. We thus propose that S100A9 deficiency in diseases in which DCs play a more prominent role will result in increased tissue inflammation. Further research is required to discriminate whether biological effects of S100A8/A9 are mediated by either intracellular versus extracellular actions or pro-inflammatory versus anti-inflammatory effects. In addition, the cell types that contribute to S100A8/A9 expression and secretion and as well as their specific roles in different disease states needs to be investigated. Answers to these issues are needed before we can more fully understand the roles of S100A8 and S100A9 in different skin diseases.