Chapter 12. Chemokines

The skin is an organ in which the migration, influx, and egress of leukocytes occur in both homeostatic and inflammatory processes. Chemokines and their receptors are accepted as vital mediators of cellular trafficking. Since the discovery of the first chemoattractant cytokine or chemokine in 1977, 50 additional new chemokines and 17 chemokine receptors have been discovered. Most chemokines are small proteins with molecular weights in the 8–10 kDa range and are synthesized constitutively in some cells and can be induced in many cell types by cytokines. Initially associated only with recruitment of leukocyte subsets to inflammatory sites,1 it has become clear that chemokines play roles in angiogenesis, neural development, cancer metastasis, hematopoiesis, and infectious diseases. This chapter will focus primarily on the function of chemokines in inflammatory conditions, but will also touch upon the role of these molecules in other settings as well.

An overview of the structure of chemokines and chemokine receptors will be provided that will detail the molecular signaling pathways initiated by the binding of a chemokine to its cognate receptor. Expression patterns of chemokine receptors will be detailed because of the many types of immune cells that potentially can be recruited to skin under inflammatory conditions. Individual chemokine receptors will be highlighted in regard to biologic function, including facilitation of migration of effector T cells into the skin and the egress of antigen-presenting cells out of the skin. Finally, the roles of chemokines and their receptors in several cutaneous diseases—atopic dermatitis, psoriasis, cancer, and infectious disease—provide a better idea of the diversity of chemokine function in skin.

Chemokines are grouped into four subfamilies based on the spacing of amino acids between the first two cysteines. The CXC chemokines (also called α-chemokines) show a C–X–C motif with one nonconserved amino acid between the two cysteines. The other major subfamily of chemokines lacks the additional amino acid and is termed the CC subfamily (or β-chemokines). The two remaining subfamilies contain only one member each: the C subfamily is represented by lymphotactin, and fractalkine is the only member of the CXXXC (or CX3C) subfamily. Chemokines can also be assigned to one of two broad and, perhaps, overlapping functional groups. One group (e.g., RANTES, MIP-1α/β LARC, etc.) mediates the attraction and recruitment of immune cells to sites of active inflammation while other (e.g., SLC and SDF-1) appear to play a role in constitutive or homeostatic migration pathways.2

The complexity and redundancy in the nomenclature of chemokines has led to the proposal for a systematic nomenclature for chemokines based on the type of chemokine (C, CXC, CX3C, or CC) and a number based on the order of discovery as proposed by Zlotnik and Yoshie.2 For example, stromal-derived factor-1 (SDF-1), a CXC chemokine, has the systematic name CXCL12. Because both nomenclatures are still in wide use, the original names (abbreviated in most cases) as well as systematic names will be used interchangeably throughout the chapter. Table 12-1 provides a list of chemokine receptors of interest in skin that are discussed in this chapter as well as the major chemokine ligands that bind to them.

Chemokines are highly conserved and have similar secondary and tertiary structure. Based on crystallography studies, a disordered amino terminus followed by three conserved antiparallel β-pleated sheets is a common structural feature of chemokines. Fractalkine is unique in that the chemokine domain sits atop a mucin-like stalk tethered to the plasma membrane via a transmembrane domain and short cytoplasmic tail.30 Although CXC and CC chemokines form multimeric structures under conditions required for structural studies, these associations may be relevant only when chemokines associate with cell-surface components such as glycosaminoglycans (GAGs) or proteoglycans. Since most chemokines have a net positive charge, these proteins tend to bind to negatively charged carbohydrates present on GAGs. Indeed the ability of positively charged chemokines to bind to GAGs is thought to enable chemokines to preferentially associate with the lumenal surface of blood vessels despite the presence of shear forces from the blood that would otherwise wash the chemokines away.

Chemokine receptors are seven transmembrane spanning membrane proteins that couple to intracellular heterotrimeric G-proteins containing α, β, and γ subunits.2 They represent a part of a large family of G-protein coupled receptors (GPCR), including rhodopsin, that have critical biologic functions. Leukocytes express several Gα protein subtypes: s, i, and q, while the β and γ subunits each have 5 and 11 known subtypes, respectively. This complexity in the formation of the heterotrimeric G-protein may account for specificity in the action of certain chemokine receptors. Normally G-proteins are inactive when GDP is bound, but they are activated when the GDP is exchanged for GTP (Fig. 12-1). After binding to a ligand, chemokine receptors rapidly associate with G-proteins, which in turn increases the exchange of GTP for GDP. Pertussis toxin is a commonly used inhibitor of GPCR that irreversibly ADP-ribosylates Gα subunits of the αi class and subsequently prevents most chemokine receptor-mediated signaling.

Activation of G-proteins leads to the dissociation of the Gα and Gβγ subunits (Fig. 12-1). The Gα subunit has been observed to activate protein tyrosine kinases and mitogen-activated protein kinase, leading to cytoskeletal changes and gene transcription. The Gα subunit retains GTP, which is slowly hydrolyzed by the GTPase activity of this subunit. This GTPase activity is both positively and negatively regulated by GTPase-activating proteins [also known as regulator of G-protein signaling (RGS) proteins]. The Gβγ dimer initiates critical signaling events in regard to chemotaxis and cell adhesion. It activates phospholipase C (PLC)32 leading to formation of diacylglycerol (DAG) and inositol triphosphate [Ins(1,4,5)P3]. Ins(1,4,5)P3 stimulates Ca2+ entry into the cytosol, which along with DAG, activates protein kinase C isoforms. While the Gβγ subunits have been shown to be critical for chemotaxis, the Gαι subunit has no known role in chemotactic migration. There is also evidence that binding of chemokine receptors results in the activation of other intracellular effectors including Ras and Rho, phosphatidylinositol-3-kinase [PI(3)K].33

RhoA and protein kinase C appear to play a role in integrin affinity changes, while PI(3)K may be critical for changes in the avidity state of LFA-1. Other proteins have been found that regulate the synthesis, expression, or degradation of G-protein coupled receptors. For example, receptor-activity-modifying proteins (RAMPS) act as chaperones of seven transmembrane spanning receptors and regulate surface expression as well as the ligand specificity of chemokine receptors (Fig. 12-1). Importantly, after chemokine receptors are exposed to appropriate ligands, they are frequently internalized, leading to an inability of the chemokine receptor to mediate further signaling. This downregulation of chemokine function, which has been termed “desensitization,” occurs because of phosphorylation of Ser/Thr residues in the C-terminal tail by proteins termed GPCR kinases (GRK) and subsequent internalization of the receptor (Fig. 12-1). Desensitization may be an important mechanism for regulating the function of chemokine receptors by inhibiting cell migration as leukocytes arrive at the primary site of inflammation.

Generally speaking, chemokines are thought to play at least three different roles in the recruitment of host defense cells, predominantly leukocytes, to sites of inflammation.34 First, they provide the signal or signals required to cause leukocytes to come to a complete stop (i.e., arrest) in blood vessels at inflamed sites such as skin. Second, chemokines have been shown to have a role in the transmigration of leukocytes from the lumenal side of the blood vessel to the ablumenal side. Third, chemokines attract leukocytes to sites of inflammation in the dermis or epidermis following transmigration. Keratinocytes and endothelial cells are a rich source of chemokines when stimulated by appropriate cytokines. In addition, chemokines and their receptors are known to play critical roles in the emigration of resident skin dendritic cells (i.e., Langerhans cells and dermal dendritic cells) from the skin to draining lymph nodes (LN) via afferent lymphatic vessels, a process that is essential for the development of acquired immune responses.

This section will be divided into three subsections. The first will introduce basic concepts of how all leukocytes arrest in inflamed blood vessels prior to transmigration by introducing the multistep model of leukocyte recruitment. The second will detail mechanisms of T cell migration, while the final subsection will focus on the mechanisms by which chemokines mediate the physiological migration of DC from the skin to regional LN.

The Multistep Model of Leukocyte Recruitment

In order for leukocytes to adhere and migrate to peripheral tissues, they must overcome the pushing force of the vascular blood stream as they bind to activated endothelial cells at local sites of inflammation. According to the multistep or cascade model of leukocyte recruitment (Fig. 12-2), one set of homologous adhesion molecules termed selectins mediates the transient attachment of leukocytes to endothelial cells while another set of adhesion molecules termed integrins and their receptors (immunoglobulin superfamily members) mediates stronger binding (i.e., arrest) and transmigration.35 The selectins (E-, L-, and P-selectins) are members of a larger family of carbohydrate-binding proteins termed lectins. The selectins bind their respective carbohydrate ligands located on protein scaffolds and thus mediate the transient binding or “rolling” of leukocytes on endothelial cells.

The skin-associated vascular selectin known as E-selectin is upregulated on endothelial cells by inflammatory cytokines such as tumor necrosis factor (TNF)-α and binds to sialyl Lewis x-based carbohydrates. E-selectin ligands form distinct epitopes known as the cutaneous lymphocyte-associated antigen (CLA). CLA is expressed by 10%–40% of memory T cells and has been suggested as a marker for skin-homing T cells.36 At least two chemokine receptors (CCR10 and CCR4) show preferential expression in CLA+ memory T cells.8,20 While E-selectin is likely to be an important component of skin-selective homing, there is also evidence to suggest that L-selectin is involved in T cell migration to skin.37,38

In the second phase of this model, leukocyte integrins such as those of the β2 family must be “turned on” or activated from their resting state in order to bind to their counter receptors such as intercellular adhesion molecule-1 (ICAM-1) that are expressed by endothelial cells. A vast array of data suggest that the binding of chemokines to leukocyte chemokine receptors plays a critical role in activating both β1 and β2 integrins.33,39 Activation of chemokine receptors leads to a complex signaling cascade (Fig. 12-1) that causes a conformational change in individual integrins that leads to increases in the affinity and avidity of individual leukocyte integrins for their ligands. Furthermore, later steps of migration (i.e., transmigration or diapedesis) have been shown to be dependent on chemokines as well in selective cases.13 In the case of neutrophils, their ability to roll on inflamed blood vessels likely depends on their expression of L-selectin and E-selectin ligands while their arrest on activated endothelia likely depends on their expression of CXCR1 and CXCR2 as described below for wound healing. Integrin activation via chemokine-mediated signals appears to be more complex in T cells, which appear to use multiple chemokine receptors, and is described in more detail below.

Chemokine-Mediated Migration of T Cells

Antigen-inexperienced T cells are termed naive and can be identified by expressing three cell surface proteins: CD45RA (an isoform of the pan-leukocyte marker), L-selectin, and the chemokine receptor CCR7. These T cells migrate efficiently to secondary LN, where they may make contact with antigen-bearing dendritic cells from the periphery. Once activated by dendritic cells presenting antigen, T cells then express CD45RO, are termed “memory” T cells, and appear to express a variety of adhesion molecules and chemokine receptors, which facilitate their extravasation from blood vessels to inflamed peripheral tissue. A specific subset of CCR7−, L-selectin memory T cells has been proposed to represent an effector memory T cell subset that is ready for rapid deployment at peripheral sites in terms of their cytotoxic activity and ability to mobilize cytokines.14

Although chemokines are both secreted and soluble, the net positive charge on most chemokines allows them to bind to negatively charged proteoglycans such as heparin sulfate that are present on the lumenal surface of endothelial cells, thus allowing them to be presented to T cells as they roll along the lumenal surface (Fig. 12-2). After ligand binding, chemokine receptors send intracellular signals that lead to increases in the affinity and avidity of T-cell integrins such as LFA-1 and VLA-4 for their endothelial receptors ICAM-1 and VCAM-1, respectively.40 Only a few chemokine receptors (CXCR4, CCR7, CCR4, and CCR6) are expressed at sufficient levels on resting peripheral blood T cells to mediate this transition. With activation and IL-2 stimulation, increased numbers of chemokine receptors (e.g., CXCR3) are expressed on activated T cells, making them more likely to respond to other chemokines. In several different systems, inhibition of specific chemokines produced by endothelial cells or chemokine receptors found on T cells dramatically influences T cell arrest in vivo and in vitro.41

CXCR3 serves as a receptor for chemokine ligands Mig, IP-10, and I-Tac. All three of these chemokines are distinguished from other chemokines by being highly upregulated by interferon-γ. Resting T cells do not express functional levels of CXCR3, but upregulate this receptor with activation and cytokines such as IL-2. Once expressed on T cells, CXCR3 is capable of mediating arrest of memory T cells on activated endothelial cells.27 The expression of its chemokine ligands is strongly influenced by the cytokine interferon-γ, which synergistically works with proinflammatory cytokines such as TNF-α to increase expression of these ligands by activated endothelial cells27 and epithelial cells.

In general, activation of T cells by cytokines such as IL-2 is associated with the enhanced expression of CCR1, CCR2, CCR5, and CXCR3. Just as Th1 and Th2 (T cell) subsets have different functional roles, it might have been predicted that these two subsets of T cells would express different chemokine receptors. Indeed, CCR49,42,43 and CCR36 are associated with Th2 cells in vitro while Th1 cells are associated with CCR5 and CXCR3.44

In some instances, chemokine receptors may be regarded as functional markers that characterize distinct T helper cell subsets while also promoting their recruitment to inflammatory sites characterized by “allergic” or “cell-mediated” immune responses, respectively. When T cells are activated in vitro in the presence of Th1-promoting cytokines, CXCR3 and CCR5 appear to be highly expressed, while in the presence of Th2-promoting cytokines, CCR4, CCR8, and CCR3 expression predominates. In rheumatoid arthritis, a Th1-predominant disease, many infiltrating T cells express CCR5 and CXCR345 whereas, in atopic disease, CCR4 expressing T cells may be more frequent.9 CCR6 has recently been described as a marker for a newly characterized T-helper subset, expressing the hallmark effector cytokines IL-17 and IL-22.46 These so-called Th17 cells play a central role in the pathogenesis of psoriasis and other chronic inflammatory autoimmune diseases.47 However, in normal skin, the majority of skin resident T cells also coexpress CCR6, suggesting that CCR6 and CCL20 interactions regulate T cell infiltration in the skin under inflammatory as well as homeostatic conditions.48

While certain chemokine receptors characterize distinct T-cell subsets, flexible regulation of their expression may increase the migratory potential of circulating T cells to diverse tissues. For example, under some conditions, both Th1 and Th2 type T cells can express CCR4.43 Similarly, T regulatory cells (Treg) and Th17 cells share chemokines receptors with other T cell lineages but may alter their chemokine receptor expression profiles, depending on the microenvironment in which they are activated.49

The epidermis is a particularly rich source of chemokines, including RANTES, MIP-3α (CCL20), MCP-1, IP-10, IL-8, LARC, and TARC, which likely contribute to epidermal T cell migration. Keratinocytes from patients with distinctive skin diseases appear to express unique chemokine expression profiles. For instance, keratinocytes derived from patients with atopic dermatitis synthesized mRNA for RANTES at considerably earlier time points in response to IL-4 and TNF-α in comparison to healthy individuals and psoriatic patients.50 Keratinocytes derived from psoriatic patients synthesized higher levels of IP-10 with cytokine stimulation as well as higher constitutive levels of IL-8,50 a chemokine known to recruit neutrophils. IL-8 may contribute to the large numbers of neutrophils that localize to the suprabasal and cornified layers of the epidermis in psoriasis. IP-10 may serve to recruit activated T cells of the Th1 helper phenotype to the epidermis and has been postulated to have a role in the recruitment of malignant T cells to the skin in cutaneous T cell lymphomas.28

CTACK/CCL27 is selectively and constitutively expressed in the epidermis, and its expression is only marginally increased under inflammatory conditions.21 Interestingly, CTACK has been reported to preferentially attract CLA+ memory T cells in vitro21 and has been demonstrated to play a role in the recruitment and function of skin-homing T cells in inflammatory disease models.51,52

Chemokines in the Trafficking of Dendritic Cells from Skin to Regional Lymph Nodes

Antigen-presenting cells, including dendritic cells (DC) of the skin, are critical initiators of immune responses and their trafficking patterns are thought to influence immunological outcomes. Their mission includes taking up antigen at sites of infection or injury and bringing these antigens to regional LN where they both present antigen and regulate the responses of T and B cells. Skin-resident DCs are initially derived from hematopoietic bone marrow progenitors53 and migrate to skin during the late prenatal and newborn periods of life. Under resting (steady state) conditions, homeostatic production by keratinocytes of CXCL14 (receptor unknown) may be involved in attracting CD14+ DC precursors to the basal layer of the epidermis.54 Similarly, Langerhans cells (LC) as well as CD1c+ LC precursors are strongly chemoattracted to keratinocyte-derived CCL20.55 Under inflammatory conditions, when skin-resident DC and LC leave the skin in large numbers, keratinocytes release a variety of chemokines, including CCL2 and CCL7 (via CCR2)4 and CCL20 (via CCR6),56 which may attract monocyte-like DC precursors to the epidermis in order to replenish the LC population.

When activated by inflammatory cytokines (e.g., TNF-α and IL-1β), lipopolysaccharide, or injury, skin DC, including LC, leave the epidermis, enter afferent lymphatic vessels, and migrate to draining regional LN where they encounter both naive and memory T cells. Chemokines guide the DC on this journey. Activated DC specifically upregulate expression of CCR7, which binds to secondary lymphoid tissue chemokine (SLC/CCL21), a chemokine expressed constitutively by lymphatic endothelial cells15,57 (eFig. 12-2.1). SLC guides DC into dermal lymphatic vessels and helps retain them in SLC-rich regional draining LN (Fig. 12-3).58

Interestingly, naive T cells also strongly express CCR7 and use this receptor to arrest on high endothelial venules.59 The importance of the CCR7 pathway is demonstrated by LC from CCR7 knockout mouse that demonstrate poor migration from the skin to regional LN16 and by the observation that antibodies to SLC block migration of DC from the periphery to LN.15 Thus, CCR7 and its ligands facilitate the recruitment of at least two different kinds of cells—naive T cells and DC—to the LN through two different routes under both inflammatory16 and resting conditions.58

After DC reach the LN, they must interact with T cells to form a so-called “immunological synapse” that is critical for T cell activation. Activated DC secrete a number of chemokines, including macrophage-derived chemokine (MDC),60 which attracts T cells to the vicinity of DC and promotes adhesion between the two cell types.61,62 CCR5 (via CCL3/4) has also been identified as mediating recruitment of naive CD8+ T cells to aggregates of antigen-specific CD4+ T cells and DC.63 Therefore, chemokines orchestrate a complex series of migration patterns bringing both DC and T cells to the confines of the LN, where expression of chemokines by DC themselves appears to be a direct signal for binding of the T cell (Fig. 12-3).

Atopic Dermatitis

Atopic dermatitis is a prototypical Th2-mediated, allergic skin disease with multifactorial genetic and environmental factors involved in its pathogenesis. Although multiple chemokines have been associated with the atopic phenotype, the roles of CCR4 and CCR10 in AD have been particularly well documented.64 Clinical data from humans as well as experimental data in the NC/Nga mouse model of atopic dermatitis suggest that the Th2-associated chemokine receptor, CCR4, in conjunction with its ligand, TARC/CCL17, may play a role in recruiting T cells to atopic skin. In patients with atopic dermatitis, CLA+CCR4+CCR10+ lymphocytes were found to be increased in the peripheral blood and in lesional skin compared to controls.9 Moreover, serum levels of TARC/CCL17 and CTACK/CCL27 in atopic dermatitis patients were significantly higher than concentrations found in healthy or psoriatic controls and correlated with disease severity.10

CCL18, whose receptor is currently unknown, has been reported to be expressed at higher levels in the skin of patients with atopic disease compared to psoriasis.65 CCL18 is produced by antigen-presenting cells and attracts CLA+ memory T cells to the skin.66 Elevated levels of CCL18 can be found in the skin and sera of patients with AD but show a significant decrease after therapy.67 Of note, CCL18 and another chemokine, CCL1 (produced by mast cells and endothelial cells), are elicited in volunteer skin after topical challenge with dust mite allergen and Staphylococcal superantigen.65,68

The recruitment of eosinophils to skin is a frequently observed finding in allergic skin diseases, including atopic dermatitis and cutaneous drug reactions, and likely is mediated by chemokines. Eotaxin/CCL11 was initially isolated from the bronchoalveolar fluid of guinea pigs after experimental allergic inflammation and binds primarily to CCR3, a receptor expressed by eosinophils,69 basophils, and Th2 cells.6 Injection of eotaxin into the skin promotes the recruitment of eosinophils while anti-eotaxin antibodies delay the dermal recruitment of eosinophils in the late-phase allergic reaction in mouse skin.70 Immunoreactivity and mRNA expression of eotaxin and CCR3 are both increased in lesional skin and serum of patients with atopic dermatitis, but not in nonatopic controls.71,72 Eotaxin has also been shown to increase proliferation of CCR3-expressing keratinocytes in vitro.73 Finally, expression of eotaxin (and RANTES) by dermal endothelial cells has been correlated with the appearance of eosinophils in the dermis in patients with onchocerciasis that experience allergic reactions following treatment with ivermectin.74 The observations above suggest that production of eotaxin and CCR3 may contribute to the recruitment of eosinophils and Th2 lymphocytes in addition to stimulating keratinocyte proliferation.

Psoriasis

Psoriasis is characterized by hyperplasia of the epidermis (acanthosis) and a prominent dermal and epidermal inflammatory infiltrate, typically resulting in thickened, hyperkeratotic plaques. The inflammatory infiltrate of psoriatic skin is predominantly composed of Th1- and Th17-polarized memory T cells, as well as neutrophils, macrophages, and increased numbers of dendritic cells.75 As shown in eFig. 12-3.1 and reviewed by others,64 there is a growing body of evidence supporting a central role for chemokines in regulating the complex events leading to psoriatic skin inflammation. Chemokines, including CCL2076 and CCL178 mediate the arrest of effector memory T cells on endothelial cells that synthesize these chemokines.77 In addition, both CCL17 and CCL20 can be synthesized by keratinocytes, possibly contributing to T cell migration to the epidermis.

While psoriasis has traditionally been considered a classical Th1-associated disease, accumulating evidence points to an important pathogenetic contribution of Th17 cells, which strongly express CCR6.79 Th17 cells, their signature effector cytokines IL-17 and IL-22, as well as high levels of IL-23, a major growth and differentiation factor for Th17 cells, are abundant in psoriatic skin lesions.80 Recent research suggests that CCR6 and its ligand, CCL20, are important mediators of psoriasis since both CCL20 as well as CLA+CCR6+ skin-homing Th17 cells are found in abundance in lesional psoriatic skin.80,81 Moreover, CCR6-deficient mice failed to develop psoriasis-like inflammation82 in response to intradermal IL-23 injections, a murine model for human psoriasis83 (eFig. 12-3.2). Interestingly, CCR6 was required for both T cell dependent as well as T cell independent skin inflammation in this model.82

Neutrophils found in the epidermis of psoriatic skin are probably attracted there by high levels of IL-8, which would act via CXCR1 and CXCR2. In addition to attracting neutrophils, IL-8 is an ELR+ CXC chemokine that is known to be angiogenic, and it may also attract endothelial cells. This may lead to the formation of the long tortuous capillary blood vessels in the papillary dermis that are characteristic of psoriasis. Moreover, keratinocytes also express CXCR2 and thus may be autoregulated by the expression of CXCR2 ligands in the skin. Of note, an IL-8/CXCL8-producing population of memory T cells that express CCR6 has been isolated from patients with acute generalized exanthematous pustulosis (AGEP), a condition induced most commonly by drugs (e.g., aminopenicillins) and characterized by small intraepidermal or subcorneal sterile pustules.84 Similar T cells have been isolated from patients with Behçet's disease and pustular psoriasis.78 It is possible that this subpopulation of T cells contributes to neutrophil accumulation in the stratum corneum (Munro's abscesses) in psoriasis and other inflammatory skin disorders characterized by neutrophil-rich infiltrates in the absence of frank infection.

Cancer

Chemokines may play a role in tumor formation and immunity in several distinct ways, including the control of angiogenesis and the induction of tumor immune responses.85 CXC chemokines that express a three-amino-acid motif consisting of glu-leu-arg (ELR) immediately preceding the CXC signature are angiogenic while most non-ELR CXC chemokines, except SDF-1, are angiostatic. Interestingly, it is not clear that ELR− chemokines actually bind to chemokine receptors in order to reduce angiogenesis. It has been proposed that they act by displacing growth factors from proteoglycans. In any event, the balance between ELR+ versus ELR− chemokines is thought to contribute to the complex regulation of angiogenesis at tumor sites. IL-8, a prototypical ELR+ chemokine, can be secreted by melanoma cells and has been detected in conjunction with metastatic dissemination of this cancer,86 which may be related to its ability to attract circulating tumor cells to primary tumors and to influence leukocyte and endothelial cell recruitment.87,88 IL-8 may also act as an autocrine growth factor for melanoma24 as well as several other types of cancer. Although CXCR1 and CXCR2 bind IL-8 in common, several other ELR+ CXC chemokines also bind to and activate CXCR2.

Tumors, including melanoma, have long been known to secrete chemokines that can attract a variety of leukocytes. The question arises as to why this is not deleterious to the tumor itself. Breast cancers, for instance, are known to secrete macrophage chemotactic protein-1 (MCP-1), a chemokine that attracts macrophages through CCR2. Higher tissue levels of MCP-1 correlate with increasing number of macrophages within the tissue. While chemokines secreted by tumor cells do lead to recruitment of immune cells, this does not necessarily lead to increased clearance of the tumor.89

Inflammatory cells such as macrophages may actually play a critical role in cancer invasion and metastasis. Firstly, MCP-1 may increase expression of macrophage IL-4 through an autocrine feedback loop and possibly skew the immune response from Th1 to Th2. Interestingly, MCP-1-deficient mice show markedly reduced dermal fibrosis following dermal challenge with bleomycin, a finding of possible relevance to the pathogenesis of conditions such as scleroderma.5 Secondly, macrophages may promote tumor invasion and metastasis.90 The antitumor effects of specific chemokines may occur by a variety of mechanisms. ELR− CXCR3 ligands such as IP-10 are potently antiangiogenic and may act as downstream effectors of IL-12-induced, NK cell-dependent angiostasis.91 Of note, some cancer cells can synthesize LARC, attracting immature DC that express CCR6.92 Experimentally, LARC has been transduced into murine tumors, where it attracts DC in mice and suppresses tumor growth in experimental systems.93 Lastly, chemokines produced by tumor cells may attract CD4+CD25+ T regulatory cells (Tregs) that suppress host antitumor cytolytic T cells.94

Tumor metastasis is the most common cause of mortality and morbidity in cancer. With skin cancers such as melanoma, there is a propensity for specific sites such as brain, lung, and liver, as well as distant skin sites. Cancers may also metastasize via afferent lymphatics and eventually reach regional draining LN. The discovery of nodal metastasis often portends a poor prognosis for the patient. In fact, the presence of nodal metastases is one of the most powerful negative predictors of survival in melanoma.95

Chemokines may play an important role in the site-specific metastases of cancers of the breast and of melanoma96 (Fig. 12-4). Human breast cancer as well as melanoma lines express the chemokine receptors CXCR4 and CCR7, whereas normal breast epithelial cells and melanocytes do not appear to express these receptors.97 CXCR4 is expressed in over 23 different solid and hematopoietic cancers. Broad expression of this receptor may be due to its regulation by hypoxia, a condition common to growing tumors, via the hypoxia inducible factor-1α transcription factor.98 Notably stromal fibroblasts within human cancers express the CXCR4 ligand, CXCL12, which stimulates tumor growth as well as angiogenesis.99 In several different animals of breast cancer97 and melanoma metastasis,29 inhibition of CXCR4 with antibodies or peptides resulted in dramatically reduced metastases to distant organs. Expression of CCR7 by cancer cells, including gastric carcinoma and melanoma, appears to be critical for invasion of afferent lymphatics and LN metastasis. CCR7-transfected B16 murine melanoma cells were found to metastasize with much higher efficiency to regional LN compared to control B16 cells after inoculation into the footpad of mice,17 but CCR7 also directly stimulates primary B16 tumor development as well.100 CCR9 may also play a role in melanoma metastasis to the small bowel, which shows high expression of the CCR9 ligand, CCL25.19

CCR10 is highly expressed by melanoma primary tumors22 and is correlated with nodal metastasis in melanoma patients101 and in experimental animal models (eFig. 12-4.1).22 Engagement of CCR10 by CTACK results in activation (via phosphorylation) of the phosphatidylinositol 3-kinase (PI3K) and Akt signaling pathways, leading to antiapoptotic effects in melanoma cells.22 Because CTACK is constitutively produced by keratinocytes, it may act as a survival factor for both primary as well as secondary (metastatic) melanoma tumors that express CCR10. In fact, CCR10-activated melanoma cells become resistant to killing by melanoma antigen-specific T cells (eFig. 12-4.1).22 Interestingly, CCR4,11 CXCR4,102 and CCR1023,103 have been implicated in the trafficking and/or survival of malignant T (lymphoma) cells to skin. Thus, a limited number of specific chemokine receptors appear to play distinct, nonredundant roles in facilitating cancer progression and metastasis (summarized in Fig. 12-4).

Infectious Diseases

Although chemokines and chemokine receptors may have evolved as a host response to infectious agents, recent data suggest infectious organisms may have coopted chemokine- or chemokine receptor-like molecules to their own advantage in selected instances. A variety of microorganisms express chemokine receptors, including US28 by cytomegalovirus and Kaposi's sarcoma herpes virus (or human herpes virus-8) G-protein coupled receptor (GPCR). In the case of KSHV GPCR, this receptor is able to promiscuously bind several chemokines. More importantly, it is constitutively active and may work as a growth promoter in Kaposi's sarcoma.104

The human immunodeficiency virus (HIV)-1, the causative agent of the acquired immunodeficiency syndrome (AIDS), is an enveloped retrovirus that enters cells via receptor-dependent membrane fusion (see Chapter 198). CD4 is the primary fusion receptor for all strains of HIV-1 and binds to HIV-1 proteins, gp120 and gp41. However, different strains of HIV-1 have emerged that preferentially use CXCR4 (T-tropic) or CCR5 (M-tropic) or either chemokine receptor as a coreceptor for entry. While other chemokine coreceptors can potentially serve as coreceptors, most clinical HIV-1 strains are primarily dual-tropic for either CCR5 or CXCR4.3

The discovery of a 32-base pair deletion (D32) in CCR5 in some individuals that leads to low levels of CCR5 expression in T cells and dendritic cells and correlates with a dramatic resistance to HIV-1 infection demonstrated a clear role for CCR5 in the pathogenesis of HIV-1 infection.105 Interestingly, the frequency of Δ32 mutations in humans is surprisingly high, and the complete absence of CCR5 in homozygotes has only been associated with a more clinically severe form of sarcoidosis. Otherwise, these individuals are healthy. In fact, there is an association of less severe autoimmune diseases in patients with these mutations.106

LC reside in large numbers in the genital mucosa and may be one of the first initial targets of HIV-1 infection.107 Since infected (activated) LC likely enter dermal lymphatic vessels and then localize to regional LN as described earlier, the physiologic migratory pathway of LC may also coincidentally lead to the transmission of HIV-1 to T cells within secondary lymphoid organs. CCR5 is expressed by immature or resting LC in the epidermis and is the target of CCR5 analogs of RANTES that block HIV infection.108 Already, an FDA-approved small molecule inhibitor of CCR5, maraviroc, is available for use in treatment of HIV disease and may show fewer adverse effects than certain reverse transcriptase inhibitors.109 CXCR4 antagonists may also be of clinical utility with T- or dual-tropic viruses.110 A newly described autosomal dominant genetic syndrome comprised of warts (human papilloma virus (HPV)-associated), hypogammaglobulinemia, infections, and myelokathexis (WHIM) is the result of an activating mutation (deletion) in the cytoplasmic tail of the CXCR4 receptor or in yet unidentified downstream regulators of CXCR4 function.111,112 Bacterial infections are common because myelokathexis is associated with neutropenia and abnormal neutrophil morphology. The nearly universal presence of HPV infections associated with this syndrome can involve multiple common, as well as genital, wart subtypes (eFig. 12-4.2) and suggest a critical role for normal CXCR4 function in immunological defense against this common human pathogen.

The skin is rich in cells (keratinocytes, fibroblasts, endothelial cells, and immune cells) that are able to produce chemokines. Chemokines not only orchestrate the migration of inflammatory cells but also play roles in angiogenesis, cancer metastasis, and cellular proliferation. Other unanticipated biologic roles may ultimately be discovered. Just two of the promising therapeutic applications of chemokines (or molecules that mimic chemokines) may be in (1) preventing undesirable migration into the skin by preventing arrest of T cells or other inflammatory cells on activated endothelium, and (2) blocking the infection of dendritic cells and T cells by HIV-1 virus using CCR5 analogs. Signaling pathways are just beginning to be understood, and further work needs to be done to understand the regulation of these receptors, the specificity of intracellular activities, and the mechanism by which chemokine receptors work in the face of multiple chemokines present in many inflammatory sites.