Intracellular activities of S100A8/A9
In the intracellular milieu, S100 proteins are considered as calcium sensors changing their conformation in response to calcium influx and then mediating calcium signals by binding to other intracellular proteins. In a mouse knock-out model chemokine-induced down regulation of the cytosolic Ca²⁺-level was detected (Nacken et al., 2005).
After calcium binding, the S100A8/A9 protein complex binds specifically polyunsaturated fatty acids. S100A8/A9 represents the exclusive arachidonic acid-binding capacity in the neutrophil cytosol (Kerkhoff et al., 1999), and participates in NADPH oxidase activation by transferring arachidonic acid to membrane-bound gp91^phox during interactions with two cytosolic oxidase activation factors, p67^phox and Rac-2. The functional relevance of S100A8/A9 in the phagocyte NADPH oxidase activation was demonstrated by the impairment of NADPH oxidase activity in neutrophil-like NB4 cells, after specifically blocking S100A9 expression, and employing bone marrow-derived PMNs from S100A9^-/- mice (Kerkhoff et al., 2005).
In accordance to their role in myeloid cells, S100A8/A9 enhances epithelial NADPH oxidases (Benedyk et al., 2007). As a consequence of enhanced ROS levels, NF-kB activation and subsequently TNF-alpha and IL-8 mRNA levels are increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kB is a redox-sensitive transcription factor. Further consequences of S100A8/A9-mediated NF-kB activation are reduced cell growth, increased expression of differentiation markers, and enhanced PARP cleavage as an indicator of increased cell death (Voss et al., 2011).
In view of the stress response-induced expression of the two S100 proteins in keratinocytes these findings have great implications for tissue remodeling and repair. For example, keratinocytes acquire an activated state after cutaneous wounding in which proliferation is favored over differentiation in order to replenish the lost material and rapidly close the site of injury. Thus, it is likely to hypothesize that S100A8/A9-mediated growth reduction is required for the upcoming cell fate decision of damaged cells, i.e. for a survival phase to be followed by differentiation, proliferation, or apoptosis. These data have also an impact on tumorigenesis since S100 gene expression is associated with neoplastic disorders.
In migrating monocytes the S100A8/A9 complex has been found to be associated with cytoskeletal tubulin and to modulate transendothelial migration (Vogl et al., 2004). Investigations using two different mouse knock-out models demonstrated no obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation.

Extracellular activities of S100A8/A9
The S100/calgranulins display antimicrobial activity by depriving bacterial pathogens of essential trace metals such as Zn²⁺ and Mn²⁺ (Steinbakk et al., 1990; Murthy et al., 1993; Clohessy and Golden, 1995; Sohnle et al., 2000). In the context of inflammation, it has been proposed that S100A8/A9 is massively released when neutrophils die to provide a growth-inhibitory type of host defense that is adjunctive to the usual microbicidal functions by binding metals other than Ca²⁺ (Corbin et al., 2008).
In addition, S100/calgranulins serve as leukocyte chemoattractants (Lackmann et al., 1992; Lackmann et al., 1993; Kocher et al., 1996; Lim et al., 2008). Murine S100A8 has potent chemotactic activity for neutrophils and monocytes in vitro and in vivo (Lackmann et al., 1992). In contrast, human S100A8 displays only weak leukocyte chemotactic activity in vitro and in vivo (Lackmann et al., 1993). Detailed analysis revealed that the hinge region contributes to the chemotactic activity of murine, but not human S100A8. These data questioned whether the proteins are orthologs since there is a high degree of homology between murine and human S100A8 but a functional divergence. Intriguingly, human S100A12 is chemotactic and the hinge region of human S100A12 has been implicated herein (Yang et al., 2001). Thus, the functional and sequence divergence suggested complex evolution of the S100 family in mammals.
The putative pro-inflammatory functions of S100A8 and S100A9 have recently been investigated in two different mouse knock-out models. S100A9 deficiency did not result in an obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation. In addition, chemokine-induced down regulation of the cytosolic Ca²⁺-level was detected. Obviously, these in vitro effects are compensated by alternative pathways in vivo.
Remarkably, cancer cells utilize S100A8 and S100A9 as guidance for the adhesion and invasion of disseminating malignant cells (Hiratsuka et al., 2006). In the context of malignancy it was reported that S100A8/A9 attracts Mac-1⁺ myeloid cells to the lung tissue. Recruited Mac-1⁺ myeloid cells in lung in turn produce S100A8/A9 in response to primary malignant cells in a so called "premetastatic phase". This phase shows the general characteristics of an inflammation state which facilitates the micro-environmental changes required for the migration and implantation of primary tumor cells to lung tissue. After preparation of the target tissue for accepting the malignant cells, tumor cells mimic Mac-1⁺ myeloid cells in response to S100A8/A9 chemotactic signaling and migrate to lung. So, it seems that tumor cells and Mac-1⁺ myeloid cells utilize a common pathway for migration to lung which involves the activation of mitogen-activated protein kinase pathway (Hiratsuka et al., 2006). These findings suggest S100A8/A9 as an attractive target for the development of strategies counteracting tumor metastasizing to certain organs.
S100A8 and S100A9 have been identified as important endogenous damage-associated molecular pattern (DAMP) proteins. Although receptors for S100A8/A9 are still largely uncharacterized, more recent findings support the notion that they function as potent ligands of pattern-recognition receptors, such as the toll-like receptor 4 (TLR4) (Vogl et al., 2007) and the receptor for advanced glycation end products (RAGE) (Srikrishna and Freeze, 2009).
The S100/calgranulins display cytokine-like functions, including activation of the receptor for advanced glycation endproducts (RAGE) (Hofmann et al., 1999; Herold et al., 2007). RAGE is a member of the immunoglobulin superfamily and present on numerous cell types. It has been shown to play crucial roles in a variety of pathophysiological situations, such as wound healing, atherosclerotic lesion development, tumor growth and metastasis, systemic amyloidosis, and Alzheimer disease (Bierhaus et al., 2005). RAGE/S100 interaction has been considered a very attractive model to explain how RAGE and its proinflammatory ligand contribute to the pathophysiology of several inflammatory diseases.
Beside the above mentioned receptors a number of other cell surface binding sites specific for S100A8/A9 have been reported, such as novel carboxylated glycans (Srikrishna et al., 2001), heparan sulfate glycosaminoglycans (Robinson et al., 2002), beta2-integrin (Newton and Hogg, 1998), and the fatty acid transporter FAT/CD36 (Kerkhoff et al., 2001). Therefore, the cell surface receptor of S100A8/A9 is still in debate.
Interestingly, the growth-stimulatory activity of S100A8/A9 has been demonstrated to be mediated by binding to the receptor of advanced glycation end products (RAGE) (Ghavami et al., 2008b; Turovskaya et al., 2008; Gebhardt et al., 2008). It is likely to speculate that the selective up-regulation of S100 proteins may be of importance for survival and proliferation of metastasizing cancer cells.
S100A8/A9 complexes that are secreted from phorbolester-stimulated neutrophil-like HL-60 cells have been shown to carry the eicosanoid precursor arachidonic acid (Kerkhoff et al., 1999). The S100A8/A9-arachidonic acid complex is recognized by the fatty acid transporter FAT/CD36, and the fatty acid is rapidly taken up (Kerkhoff et al., 2001). Endothelial cells as well as neutrophils themselves utilize both endogenous and exogenous arachidonic acid for transcellular production of eicosanoids (Sala et al., 1999). Therefore, the secreted S100A8/A9-AA complex may serve as a transport protein to move AA to its target cells. This may represent a mechanism by which AA-derived eicosanoids are synthesized in a cooperative manner between different cell species due to environmental cues.
S100A8/A9 displays apoptosis-inducing activity against various tumor cells (Yui et al., 1995; Yui et al., 2002; Ghavami et al., 2004; Ghavami et al., 2008a; Kerkhoff and Ghavami, 2009; Ghavami et al., 2009; Ghavami et al., 2010). It was speculated that this activity was due to the ability to bind divalent metal ions including Zn²⁺, Mn²⁺ and Cu²⁺ at sites that are distinct from Ca²⁺-binding sites. However, a number of recent reports now indicate that S100A8/A9 exerts its activity by both chelation of trace metal ions such as Zn²⁺ and cell surface receptor mediated pathways.
Although a number of receptors have been shown to bind S100A8/A9, the nature of the receptor involved in S100A8/A9-induced cell death remains to be elucidated. Experiments with certain cell lines either deficient for or over expressing components of the death signaling machinery as well as RAGE gene silencing and blocking RAGE-specific antibody approaches excluded both RAGE and the classical death receptor to be involved in S100A8/A9-induced cell death, even though S100A8/A9 can specifically bind to cancer cells and RAGE mediates the growth-promoting activity obvious at low micromolar concentrations of S100A8/A9. Clearly, investigations to identify the receptor involved in S100A8/A9-induced cell death are critical.