**3.2 Keratinocytes and chemokines**

Keratinocytes produce many different types of chemokines involved in the recruitment of immune cells in the skin. For this reason epidermal cells can play a fundamental role in collecting all the immune cells which are implicated in the beginning of the cutaneous inflammation process that characterize psoriatic disease. Specifically, keratinocytes release IL-8 (CXCL8) and related chemokines which are responsible for the intra-epidermal collection of neutrophils and so to the formation of subcorneal microabscesses, a characteristic feature of psoriasis (Nickoloff & Turka, 1994); CCL2 (MCP-1), CCL5 (RANTES), CXCL10 (IP-10), and other CXCR3 ligands are responsible to attract predominantly monocytes and Th1 cells, (Gillitzer et al., 1993; Gottlieb et al., 1998), whereas CCL20 (MIP-3α) recruits immature Langerhans cells, dendritic cells, and CLA+ T cells (Dieu-Nosjan et al., 2000; Homey et al., 2000) (Tab. 2).


Table 2. Roles of chemokines produced by psoriatic keratinocytes.

#### **3.3 Keratinocytes and other products**

Psoriatic keratinocytes are a reservoir of inflammatory mediators. Under the influence of pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-23, and IL-17, keratinocytes express a plethora of mediators, not only cytokines, thereby contributing to amplifying the inflammatory response implicated in the pathogenesis of psoriasis (Albanesi et al., 2005). Apart from pro-inflammatory cytokines as IL-1α, IL-1β, IL-6, IL-15, IL-18 and IL-20 psoriatic keratinocytes are able to produce other important factors involved in the development of the psoriatic process like vascular endothelial growth factor (VEGF) and CD1d (Tab. 3).


Table 3. Roles of VEGF and CD1d in psoriasis.

#### **3.3.1 Keratinocytes and VEGF**

18 Psoriasis

reciprocal interactions between epithelial cells (keratinocytes) and T-lymphocytes. The concomitant T-lymphocyte activation may be dependent on IL-7, and therefore the subsequent events driving toward the clinical expression and persistence of psoriasis may be IL-7 mediated (Bonifati et al., 1997). All these findings suggest an involvement of IL-7 in psoriasis, although further studies are warranted to elucidate the exact role of this molecule

Keratinocytes produce many different types of chemokines involved in the recruitment of immune cells in the skin. For this reason epidermal cells can play a fundamental role in collecting all the immune cells which are implicated in the beginning of the cutaneous inflammation process that characterize psoriatic disease. Specifically, keratinocytes release IL-8 (CXCL8) and related chemokines which are responsible for the intra-epidermal collection of neutrophils and so to the formation of subcorneal microabscesses, a characteristic feature of psoriasis (Nickoloff & Turka, 1994); CCL2 (MCP-1), CCL5 (RANTES), CXCL10 (IP-10), and other CXCR3 ligands are responsible to attract predominantly monocytes and Th1 cells, (Gillitzer et al., 1993; Gottlieb et al., 1998), whereas CCL20 (MIP-3α) recruits immature Langerhans cells, dendritic cells, and CLA+ T cells

IL-8 (CXCL8) Intra-epidermal recruitment of neutrophils

CCL20 (MIP-3α) Recruitment of dendritic cells, CLA+ T

Psoriatic keratinocytes are a reservoir of inflammatory mediators. Under the influence of pro-inflammatory cytokines such as IFN-γ, TNF-α, IL-23, and IL-17, keratinocytes express a plethora of mediators, not only cytokines, thereby contributing to amplifying the inflammatory response implicated in the pathogenesis of psoriasis (Albanesi et al., 2005). Apart from pro-inflammatory cytokines as IL-1α, IL-1β, IL-6, IL-15, IL-18 and IL-20 psoriatic keratinocytes are able to produce other important factors involved in the development of the

psoriatic process like vascular endothelial growth factor (VEGF) and CD1d (Tab. 3).

VEGF Stimulation of angiogenesis, enhancement

CD1d Activation of CD161+ NK T cells and their

Recruitment of monocytes and Th1 cells

cells and immature Langherans cells

of vascular permeability, induction of keratinocytes hyperproliferation in an

autocrine manner.

stimulation to secrete IFN-γ.

in the cytokine network of psoriasis pathogenesis.

(Dieu-Nosjan et al., 2000; Homey et al., 2000) (Tab. 2).

**Chemokines Roles**

**3.3 Keratinocytes and other products** 

Table 2. Roles of chemokines produced by psoriatic keratinocytes.

**Factors Functions**

Table 3. Roles of VEGF and CD1d in psoriasis.

**3.2 Keratinocytes and chemokines** 

CCL-2 (MCP-1) CCL5 (RANTES) CXCL10 (IP-10)

The typical erythema of psoriatic lesions is due to the increased, dilated, and tortuous capillaries that extend between the epidermal columns protruding into the dermis. The formation of new blood vessels starts with early psoriatic changes and disappears with disease clearance. Several angiogenic mediators like VEGF, hypoxia inducible factors, angiopoietins and pro-angiogenic cytokines, such as TNF-α, IL-8 and IL- 17, are involved in psoriasis development (Heidenreich et al., 2009). Interestingly, already in uninvolved, nonlesional skin significant over-expression of several VEGF isoforms was observed in patients as compared to healthy skin of normal volunteers (Henno et al., 2009). These findings suggest that angiogenesis is also one of the key features in the pathogenesis of psoriasis and various recent studies focused on the identification and role of pro-angiogenic mediators in psoriatic skin. In general, angiogenesis is tightly regulated by a balance between pro- and anti-angiogenic mediators (Heidenreich et al., 2009). VEGF, hypoxia-inducible factor-1α (HIF-1α), TNF-α, IL-8 and angiopoietins are considered to be the main players responsible for the increased vessel formation in psoriasis (Creamer et al., 2002; Heidenreich et al., 2008). Interestingly, several small molecules as well as modern biologics used for systemic therapy of psoriasis have been shown to provide not only immune regulatory effects but also influence endothelial cell biology (Heidenreich et al., 2008). Thus, direct targeting of angiogenesis could help both to dissect psoriasis pathogenesis and to develop new therapeutic strategies for psoriasis treatment by blocking angiogenic pathways driving cutaneous inflammation. Strongly increased production of VEGF by keratinocytes has been found in psoriasis (Detmar et al., 1994). Furthermore, over-expression of VEGF in the epidermis of mice triggered sub-epidermal angiogenesis and increased leukocyte adhesion to these vessels (Detmar et al., 1998), and later in life, these animals develop hyperkeratotic skin lesions with a resemblance to psoriasis (Xia et al., 2003). VEGF signaling often represents a critical rate-limiting step in physiological angiogenesis (Ferrara et al., 2003). Under physiological conditions, VEGF promotes growth of endothelial cells (ECs) derived from arteries, veins and lymphatic vessels. VEGF delivery also induces lymphoangiogenesis in mice and it is known to be a survival factor for endothelial cells both in vitro and in vivo. However, VEGF is also known as a vascular permeability factor, based on its ability to induce vascular leakage. In the meantime it is well established that such permeability enhancing activity underlies significant roles of this molecule in inflammation and other pathological circumstances (Ferrara et al., 2003). Besides its potential role in causing aberrant angiogenesis and vascular leakage in the upper dermis, VEGF may also contribute to keratinocyte proliferation and epidermal barrier homeostasis (Elias et al., 2008; Heidenreich et al., 2009). In psoriatic skin, the VEGF receptors VEGFR-1 and -2 are detectable and functional in keratinocytes (Man et al., 2006). As VEGF is secreted by keratinocytes and induces VEGFR expression in the same cells, VEGF may also contribute to keratinocyte hyperproliferation in psoriasis in an autocrine manner. This could be relevant when psoriasis is triggered by external injury (Koebner phenomenon) and interestingly disruption of the epidermal barrier homeostasis induces VEGF expression (Elias et al., 2008). Further evidence for a role of VEGF in keratinocyte proliferation comes from transgenic mice deficient in epidermal VEGF: these animals have delayed permeability barrier recovery after acute perturbation, decreased density of dermal blood vessels and lack epidermal hyperplasia as well as angiogenesis in response to sustained barrier disruption (Elias et al., 2008). Thus, physiological production of VEGF contributes to normal proliferation,

Pathogenesis of Psoriasis: The Role of Pro-Inflammatory Cytokines Produced by Keratinocytes 21

parakeratotic layer juxtaposed to the stratum corneum. CD161-positive T cells were frequently observed in direct contact with keratinocytes expressing CD1d in psoriatic plaques. Given this anatomical juxtaposition, it is possible for various types of glycolipids in the psoriatic scale to be directly exposed to the abundant keratinocyte cell surface CD1d. Moreover, given the large hydrophobic binding pockets in CD1d, the presence of CD1d on the outer layers of epidermis in psoriatic plaques opens up the possibility that various glycolipids present in the stratum corneum could play a role in triggering a response by NK-T cells or other T cell subsets capable of recognizing such glycolipids in the context of CD1d. During epidermal differentiation keratinocytes produce different amounts and types of various glycolipids, including glucosylceramides (Holleran et al., 1993). Alterations in these glycolipids in the stratum corneum can have a significant impact on the barrier function of skin. However, it is also clear that barrier perturbation can initiate cytokine cascades and thus influence inflammatory and mononuclear cell activation (Nickoloff & Naidu, 1994). A cycle can be envisioned in which pathogenic NK-T cells initiate barrier abnormality, which, in turn, would generate glycolipids that could be presented by keratinocyte CD1d and further activate CD161+ T cells in psoriasis (Kalish et al., 1994). Taken together, these findings support the idea that NK-T cells may play an important patho-physiological role in psoriasis. Besides the ability of keratinocytes to initiate (Barker et al., 1991), perpetuate (Nickoloff & Turka, 1994), and terminate (Guttierrez-Steil et al., 1998) immune reactions involving conventional T cell responses to nominal antigens and super-antigens, CD1d expression may also imbue the keratinocyte with the capacity to interact with NKR-bearing T cells. As a member of a non-classical, MHC independent, antigen-presenting system, CD1d expression as seen in psoriasis provides a novel opportunity for therapeutic targeting and for understanding the immunologic and genetic basis of psoriasis as well as the potential role for innate

The pathogenesis of psoriasis is considered to be an immunologically mediated process that takes place upon a favourable genetic background. According to this view, the presence of a yet unknown (auto)-antigen causes the generation of effector T-cells that infiltrate the skin and initiate the inflammatory process. Over its course, cutaneous infiltration of various immune cell populations and, subsequently, an activation of numerous immune and tissue cells in the skin takes place. Two fundamentally different cell types interact in the formation of a psoriatic lesion: epidermal keratinocytes and mononuclear leukocytes. Whereas keratinocytes might be viewed only as bystander cells in terms of immune activation, it is more likely that they are active participants in the recruitment and activation of leukocytes in psoriatic lesions: the interplay between keratinocytes and immune cells can be considered the main feature of the psoriasis pathogenesis. In facts whatever the sequence of events that leads to the induction of the mentioned cytokines and mediators in epidermal keratinocytes, it is highly likely that they significantly contribute to the typical changes observed in psoriatic lesions; cytokine or growth factor secretion by epidermal keratinocytes can be sufficient to recruit immune cells into the skin and induce a hyperplastic epidermis with hyperkeratosis and reproduce features of psoriatic disease. Regulation of the inflammatory events initiated or perpetuated by keratinocytes could so represent an important strategy for

the treatment of psoriasis and other chronic inflammatory skin diseases.

immunity in psoriasis (Nickoloff, 1999a, 1999b).

**4. Conclusion** 

differentiation and function of the epidermis (Heidenreich et al., 2009). Consequently, VEGF over-expression in psoriasis might contribute to the epidermal changes observed in this disease. Although immune cells are also able to secrete VEGF, the findings of VEGF overexpression in psoriatic epidermis together with the data reported from the transgenic animals strongly suggest that VEGF derived from epidermal keratinocytes acts as a key cytokine driving angiogenesis in psoriasis and as a central paracrine growth factor contributing to the pathology seen in psoriasis.

#### **3.3.2 Keratinocytes and C1d**

The expression of CD1d by normal human skin and its pronounced over-expression in psoriatic skin lesions is well documented (Bonish et al., 2000) as well as the presence of NK-T cells in the epidermis of acute and chronic psoriatic plaques (Nickoloff & Wrone-Smith, 1999; Nickoloff et al., 2000). A hallmark of NK-T cells is their expression of certain C-type lectin NK cell receptors (NKRs)4 such as CD94 and CD161. Classical NK-T cells may plan an immunoregulatory role for recognition of both self and foreign antigens and are implicated in the pathogenesis of autoimmune and inflammatory diseases like psoriasis. An important clue to the function of NK-T cells is provided by their interaction with professional antigen presenting cells (APCs) via CD1d (Huang et al., 1999). CD1d has some similarities in structure to the major histocompatibility complex class II (MHC II) molecules. While initially CD1d was believed to bind and present peptide antigens to T cells (Castano et al., 1995), more recent studies highlight its ability to present glycolipids and GPI-linked proteins (Huang et al., 1999). NK-T cells can become activated in a CD1drestricted fashion with subsequent proliferation and cytokine production, including IFN-γ and IL-4. Keratinocytes in vitro and in vivo synthesize and express CD1d, which is capable of triggering CD161+ NK-T cells to produce high levels of IFN-γ, but not IL-4. The stimulation by CD1d of T cells bearing NK receptors preferentially induces a cytokine switch to IFN-γ (Arase et al., 1996, 1997). Moreover, the differential induction of IFN-γ production, but not IL-4, after the NK-T cell clones recognized CD1d on keratinocytes has potentially important implications for psoriasis. Not only is there over-expression of CD1d by psoriatic epidermal keratinocytes and the presence of NK-T cells bearing CD94 and CD161, but the cytokine IFN-γ has been shown to trigger psoriatic lesions (Fierlbeck et al., 1990). Therefore a positive feedback loop could be established in skin due to the presence of NK-T cells being activated to produce IFN-γ upon contact with CD1d-positive keratinocytes, leading to further CD1d expression and subsequent NK-T cell release of more IFN-γ. The lack of a proliferative response by NK-T cells to CD1d keratinocytes is also consistent with the general number and distribution of CD94- and CD161-positive NK-T cells in psoriasis. Thus, the NK-T cells are never observed in tight clusters or in very large numbers as might be expected if they were undergoing a local proliferative response; rather, they are found as more evenly distributed single cells throughout a psoriatic plaque. In normal human skin CD1d is generally restricted to the outermost keratinocyte layers in the stratum granulosum just beneath the lipid-rich stratum corneum. In addition to epidermal keratinocytes, CD1d is detected on upper dermal dendritic cells, endothelium, eccrine ducts, acrosyringium, and the pilo-sebaceous unit, except for the dermal papillae and hair matrix cells. In psoriatic plaques CD1d expression was increased compared with that in normal and symptomless skin, beginning in the supra-basilar layer and extending to the outermost keratinocytes immediately beneath the parakeratotic layer juxtaposed to the stratum corneum. CD161-positive T cells were frequently observed in direct contact with keratinocytes expressing CD1d in psoriatic plaques. Given this anatomical juxtaposition, it is possible for various types of glycolipids in the psoriatic scale to be directly exposed to the abundant keratinocyte cell surface CD1d. Moreover, given the large hydrophobic binding pockets in CD1d, the presence of CD1d on the outer layers of epidermis in psoriatic plaques opens up the possibility that various glycolipids present in the stratum corneum could play a role in triggering a response by NK-T cells or other T cell subsets capable of recognizing such glycolipids in the context of CD1d. During epidermal differentiation keratinocytes produce different amounts and types of various glycolipids, including glucosylceramides (Holleran et al., 1993). Alterations in these glycolipids in the stratum corneum can have a significant impact on the barrier function of skin. However, it is also clear that barrier perturbation can initiate cytokine cascades and thus influence inflammatory and mononuclear cell activation (Nickoloff & Naidu, 1994). A cycle can be envisioned in which pathogenic NK-T cells initiate barrier abnormality, which, in turn, would generate glycolipids that could be presented by keratinocyte CD1d and further activate CD161+ T cells in psoriasis (Kalish et al., 1994). Taken together, these findings support the idea that NK-T cells may play an important patho-physiological role in psoriasis. Besides the ability of keratinocytes to initiate (Barker et al., 1991), perpetuate (Nickoloff & Turka, 1994), and terminate (Guttierrez-Steil et al., 1998) immune reactions involving conventional T cell responses to nominal antigens and super-antigens, CD1d expression may also imbue the keratinocyte with the capacity to interact with NKR-bearing T cells. As a member of a non-classical, MHC independent, antigen-presenting system, CD1d expression as seen in psoriasis provides a novel opportunity for therapeutic targeting and for understanding the immunologic and genetic basis of psoriasis as well as the potential role for innate immunity in psoriasis (Nickoloff, 1999a, 1999b).
