Lecture _: Archietective of Immune Response I Weissman



Chemokines In Inflammation

I. INTRODUCTION

Fig. 1. Chemokine and chemokine receptor diversity. The chemokines that can bind to each receptor (indicated as circles) is shown. The chemokine receptor GPR-2 is now called CCR10, a receptor for both CTACK and for the mucosal epithelial chemokine MEC (CCL28).

Chemotactic cytokines or chemokines constitute a large family of highly conserved proteins that act through G-coupled receptors to regulate diverse biological processes, including hematopoiesis, leukocyte trafficking and organogenesis. In addition, there are some situations in which medically important pathogens, including HIV-1, exploit or subvert the chemokine system. Thus, chemokines are both beneficial in host defense against infectious agents and potentially harmful in disease; however, actual clinical roles in these areas are only recently emerging. This lecture will focus on the role of chemokines in directing the trafficking of leukocytes, both in normal immunity as well as in pathologic inflammation. We will also discuss the potential for targeting specific chemokines and chemokine receptors for therapeutic utility in both inflammation and infectious disease.

II. WHAT ARE CHEMOKINES?

Fig. 2. Three-dimensional structure of a prototype CC chemokine. The monomeric structure of chemokines are very similar and comprise a critically important NH2-terminus, three anti-parallel (-strands forming a “Greek key” structure connected by loops and a COOH-terminal (-helix (from Sayle & Milner-White, Trends Biochem Sci. 20:374, 1995).

Chemokines are single polypeptide chains of 70-100 amino acids (molecular weight: 8 to 14 kDa) with a unique, highly conserved pattern of cysteines in the primary sequence (Fig. 2). The two main subfamilies, CC and CXC, are distinguished according to the position of the first two cysteines, which are adjacent (CC) or separated by one amino acid (CXC). Two additional chemokines do not fit this scheme, the C chemokine lymphotactin, and the membrane-anchored CX3C chemokine fractalkine/neurotactin (see Figures 1-4).

Chemokines bind and signal through a subfamily of G-protein-coupled receptors (GPCRs) (Figure 3). Many of the receptors are shared (i.e. they bind more than one chemokine), though some are ligand specific (i.e. they bind only one chemokine). Despite considerable promiscuity in the binding of ligands by many of the chemokine receptors, receptor specificity usually does not cross C/CC/CXC/CX3C boundaries and therefore have been grouped accordingly. Six CXC receptors (CXCR1-6) and eleven CC receptors (CCR1-11) have been identified along with the lymphotactin (XCR-1) and fractalkine/ neurotactin (CX3CR-1) receptors. There are also a number of “orphan” receptors that have significant similarity to chemokine receptors, but for which the ligands and biological functions are not known (Figure 1).

Fig. 3. Chemokine receptor-signaling pathways. Chemokine receptors are coupled to heterotrimeric G-proteins or GPCRs. These receptors contain seven transmembrane regions giving rise to three extracellular loops and three intracellular loops. Signaling is accomplished though coupled to heterotrimeric G((( proteins. Upon chemokine binding, GTP replaces GDP on G(; the G trimer dissociates, and G( and G(( independently activate downstream effectors such as adenylate cyclase, phospholipase C, phosphatidylinositol-3-kinase small GTPases of the rho family involved in cell shape and motility, and protein tyrosine kinases, which ultimately trigger events such as adhesion and chemotaxis.

There are examples of highly restricted receptor usage. For example, BLC binding only to CXCR5. However, some chemokines are highly promiscuous in receptor usage, such as RANTES which signals through CCR1, CCR3 and CCR5 (Fig. 1).

The tables below give an overview (simplified) of a few key chemokines and briefly outline the receptor usage, responsive cells and in vivo function of these chemokines and receptors

[pic]

Fig. 4 (8.13 Parham). Properties of some chemokines. Chemokines are often divided into two major groups based on whether there are one (CXC) or no amino acids (CC) between the first two cysteine residues. Lymphotactin and fractalkine are the sole representatives of two additional families of C and CX3C chemokines, respectively.

Additional chemokines discussed in this lecture:

produced attracts putative

class name receptor by cells effect

CXC BLC CXCR5 B cell follicle B cells organization of

B cell follicles

Ag-activated T cells recruitment of T cells

(T follicular helpers) for B cell help

CC TECK CCR9 thymus CD4-CD8- T cells T cell development

gut β7+ memory T cells* recruitment of T cells

to mucosal tissue

CC CTACK CCR10 keratinocytes CLA+ skin-homing recruitment of CLA+

memory T cells T cells to skin

to mucosal tissue

CC MEC CCR10 mucosal epithelium IgA plasmablasts recruitment of IgA

Plasma cells

CHEMOKINES PLAY A KEY ROLE IN IMMUNE SURVEILLANCE AND INFLAMMATION.

Lymphocyte recirculation is the process whereby lymphocytes or other leukocytes undergo repetitive cycles of migration from the blood into the tissue and back into the vasculature. T cells and B cells NK undergo continuous recirculation even in the absence of injury or inflammation as part of normal immune surveillance. (See Lecture 5). Superimposed on this is ‘recruitment’ of immune cells to inflammatory sites. The anatomic location and the nature of the inflammatory stimulus determine which leukocytes migrate to an inflammatory site; recruitment includes cells that do not recirculate such as neutrophils, eosinophils and monocytes, as well as some effector T cells.

Chemokines and their receptors are generally accepted to be central regulators of leukocyte recruitment. Many publications continue to quote the early notion that the CC receptors effect the migration of monocytes, eosinophils, basophils, and T cells, whereas CXCR1 and CXCR2 (IL-8 receptors) effect the migration of neutrophils. In fact, this early notion that all of the CXC ligands are poor lymphocyte chemoattractants turns out to be false; CCR7 ligands are robust attractants for naive B and T cells and for most circulating memory T cells, CXCR3 ligands are chemotactic for many memory and activated T cells and NK cells and IgG plasmablasts, and CXCR4 ligands attract most but not all leukocyte types, CXCR5 ligands are chemoattractants for subsets of lymphoid organ-associated (central memory) T cells and for almost all B cells through the stage of differentiation into antibody secreting cells, etc.

I. BASIC MECHANISMS OF LEUKOCYTE RECRUITMENT

As discussed previously and shown in below in Figure 3, recruitment of leukocytes into the tissue is the result of sequential, independently regulated receptor-ligand interactions. Each step potentially requires unique receptor-ligand pair interactions. Thus far, only the selectin family (L-, P- and E-selectin) and the (4 integrins have been shown to mediate initial contact (tethering) of cells flowing in the circulation. Interaction of the cell with these receptors results in the cell ‘rolling’ along the vessel wall. This is followed by rapid (within seconds) ‘triggering’ of integrin-mediated arrest, in which the cell stops rolling. This arrest involves pertussis-toxin-sensitive G(i protein-linked receptors. This arrest is spontaneously reversible (in minutes) unless further signals lead to transmigration through the endothelium followed by migration into the surrounding tissue.

Fig. 5. The multistep model of lymphocyte-endothelial cell recognition and recruitment of lymphocytes from the blood (adapted from Butcher and Picker, Science 272:60-6, 1996)

The physiologic ‘triggers’ of integrin activation are now believed to be chemokines in most instances, although other GPCR ligands can also mediate this event. A subset of chemokines trigger integrin-mediated adhesion to the endothelium under fluid flow conditions. Many chemokines display specific, low affinity binding to matrix glycosaminoglycans (GAGs) through basic residues on their non-receptor-binding face. This helps confine and focus chemokine display on the endothelium. Chemokines are also thought to provide gradients or ‘trails’ providing navigational orientation within the tissue.

V. SOURCES AND TARGETS OF CHEMOKINES

Since chemokines are involved in directing cellular trafficking, clearly the location of expression and/or presentation of chemokines are critical for their function. Most chemokines are produced by multiple cell types, but some are produced by only one or two cell types. Some are produced constitutively, others must be induced, and some are produced both constitutively and inductively, depending on the cell type. Many are up-regulated by pro-inflammatory cytokines such as tumor necrosis factor (TNF-() and interleukin-1 (IL-1) while others are up-regulated specifically by interferon gamma (IFN-(). So how does it all come together to direct leukocyte trafficking in vivo?

A leukocyte at a given point in its maturation or activation status expresses a specific set of chemokine and homing receptors. A given tissue, such as an allergic lung or patch of skin recently exposed to allergen, or an arthritic joint in a patient with rheumatoid arthritis, expresses a unique set of vascular addressins (adhesion molecules) and chemokines. The tissue is able to recruit the subset of circulating cells bearing the set of adhesion and chemokine receptors that ‘match’ the available adhesion ligands and chemokines. Clearly, since each of the receptors and ligands can be selectively expressed on leukocytes and endothelial cells, the number of possible combinations is very large – leading to the exquisite specificity of leukocyte trafficking and recruitment observed in vivo.

The bottom line is that the key to selective recruitment of leukocytes is their binding to a particular combination of receptors for selectins, integrins, and chemokines (a sort of molecular zip code). Subsequently, exposure to chemokine gradients allows precise navigation and orientation of the leukocytes within the tissue.

As for recruitment from the blood, cell localization within tissues can also be combinatorially determined….in this case by the particular combination of chemokine recpeotrs the cell expresses. In an tissue where multiple ligands are expressed by different cells or microenvironments , cells can engage one attractant gradient after another, depending on their receptor repertoire. This allows step by step control of positioning in tissues, and permits refined control of microenvironmental localization.

VI. THE IMPORTANCE OF CHEMOKINES IN CELL RECRUITMENT: AN EXAMPLE

Dust, pollens, and other allergic triggers cause epithelial cells of the respiratory tract to produce eotaxin. Eotaxin binds to the CCR3 receptor on Th2 cells recruiting them to this tissue. Th2 cells in turn produce IL-4 and IL-5, and amplify the original effects on epithelial cells (and mast cells) resulting in production of more eotaxin. Eotaxin then recruits and stimulates degranulation of eosinophils and basophils and stimulates mast cells. Recruited eosinophils and Th2 cells produce additional IL-4, IL-5 and eotaxin, which perpetuate the cycle (Fig. 6).

Fig. 6. Eotaxin is a CC-chemokine that plays a critical role in the development of allergic disease. Allergen-induced triggers cause epithelial cells to produce eotaxin. Eotaxin recruits Th2 cells that in turn produce IL-4 and IL-5, and amplify the original effects on epithelial cells (and mast cells) resulting in production of more eotaxin. Eotaxin then recruits and stimulates degranulation of eosinophils and basophils and stimulates mast cells. Recruited eosinophils and Th2 cells produce additional IL-4, IL-5 and eotaxin, which perpetuate the allergic response. (Actually, CCR4 and its ligands TARC and MDC might be a better example of attractant implicated in amplifying Th2 responses.

Fig. 7. Interferon gamma-induced chemokines play a key role in Th1-type responses: chemokine participate in amplification loops. IFN-( is a product of activated NK cells and Th1 T cells. IFN-( upregulates IP-10, MIG and ITAC in monocytes and endothelial cells resulting in recruitment of both NK and Th1 T cells – providing a second example of central involvement of chemokines in recruitment and maintenance of immune responses.

Monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-(-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that all specifically bind to a common receptor, CXCR3 (Fig. 7). As their names suggest, all are induced by interferon gamma (IFN-(), a Th1-type proinflammatory cytokine. CXCR3 is expressed by almost all lymphocytes (effector Th1 cells, NKT cells NK cells) that express IFNg. Thus, upregulation of these chemokines by IFN-( may be an important mechanism for selective recruitment of activated/effector cells which can amplify the INFg/Th1 response by further recruitment of IFNg producing cells.

It is important to note that there are many overly simplified paradigms in the literature. One is that CXCR3 is selective for Th1 cells. In fact the situation is much more complex. Although almost all Th1 cells express CXCR3, so do many IL4/IFNg co-producing cells and even some Th2 cells, as well as IgG plasmablasts. Similarly, CCR4 is expressed by almost all IL4 producing T cells, but as many Th1 as Th2 cells (in absolute numbers ) also express it, and it also functions as a diferetiating homing recpetor for systemic sites (esp skin) vs. instestines (where there are few detectable CCR4+ T cells). We will also mention the fact that CCR7 is on all naïve, but also on most memory and the majority of effector memory cells as well; again the truth in conflict with widely held beliefs. (see also below)

VII. CHEMOKINES AND HOMEOSTASIS

Chemokines are often thought of as “proinflammatory cytokines”; however, it is increasingly clear that chemokine-receptor interactions are also critical for homeostatic functions within the immune system. Chemokines, for example, are important for establishing and maintaining the complex architecture of secondary lymph nodes. Here we will focus on the chemokine ‘map’ in the secondary lymphoid tissue that directs trafficking/recruitment and microenvironmental localization of the three major participants in acquired immune responses: dendritic cells, T cells and B cells (Fig. 8).

Fig. 8. Chemokines provide a map of the lymph node and direct trafficking of T cells, B cells and dendritic cells.

Antigen-bearing, mature dendritic cells, which express CCR7, migrate into the lymph nodes and localize in the T cell zones. Dendritic cells first localize in these zones by the interaction of CCR7 with SLC/6Ckine expressed by the afferent lymphatics, and then by the interaction of CCR7 with SLC/6Ckine and ELC/MIP-3( expressed in the T cell zone. SLC/6Ckine, which is expressed on the high endothelial venue (HEV), also mediates recruitment of naïve T cells, which express CCR7, within HEV. This triggers firm adhesion of naive T cells within the HEV. Thus, the CCR7 ligands help direct migration of mature DCs and naive T cells to the T cell zone to enhance DC/T cell interaction.

BLC is expressed by stromal cells in B cell follicles and acts to recruit and organize B cells into follicles. Antigen binding by T cells upregulates CXCR5 (and down-regulates CCR7) and promotes T cell migration into the B cell follicle where they to help initiate T–dependent antibody responses

Thus, the CCR7 and CXCR5 ligands play key roles in the recruitment and organization of T, B and dendritic cells within the lymphoid organs.

Immature dendritic cells

express:

CCR1, CCR5, CCR6 and CXCR1

Mature dendritic cells

express:

CCR7

Fig. 9. Maturation of dendritic cells results in profound changes in responsiveness to chemokines.

Immature DC in the tissue express inflammatory chemokine receptors, including CCR1, CCR5, CCR6 and CXCR1, which may participate in DC recruitment to inflamed tissues (Fig. 9, above). Upon stimulation by TNF, CD40L or LPS they lose sensitivity to the ligands for CCR1, CCR5, CCR6, CXCR1, and upregulate CCR7 expression and function.

As suggested above, chemokines appear to play a role in the antigen delivery side of this equation as well. Dendritic cells arise from bone marrow progenitors and migrate via the blood to peripheral tissues where they reside as “immature” sentinels specialized for antigen capture but are unable to stimulate T cells. Upon antigen uptake and stimulation by LPS, TNF or CD40L, they undergo a maturation process marked by upregulation of molecules required for T cell activation and gain the ability to migrate to the CCR7 ligands SLC/6Ckine and ELC/MIP-3(. SLC/6Ckine is expressed by the endothelial cells lining the lymphatic vessels leading into the lymph node, and both SLC/6Ckine and the alternative CCR7 ligand MIP-3( are expressed in the T cell zone of lymph nodes and spleen suggesting a key role in recruitment of mature DC to the T cell zone. In fact, the dendritic cells in mice deficient in CCR7 ligands fail to migrate into the draining lymph nodes upon antigen capture and stimulation.

Thus, SLC/6Ckine (CCL21) appears to play a key role in the recruitment of both naive T cells and antigen-bearing mature dendritic cells to the specialized microenvironment required for T cell priming.

VIII. SPECIFIC MEMORY T CELL SUBSETS TRAFFIC SELECTIVELY

Unlike naive T cells, which recirculate homogeneously (non-selectively) through secondary lymphoid tissue, the homing behavior of memory/effector T cells is extremely heterogeneous. Distinct memory/effector subsets display restricted, often tissue-selective patterns of recirculation. In addition, memory/effector T cells can enter and recirculate through extralymphoid immune effector sites, such as the intestinal lamina propria, inflamed skin, joints, and the pulmonary interstitium.

The tissue-selective trafficking of memory/effector T cell subsets is partially explained by expression of adhesion molecules, such as cutaneous lymphocyte antigen (CLA) on skin-homing T cells and a4b7 integrins on gut-homing T cells (Figure 10, below).

It was long hypothesized but only recently shown that specific chemokine receptors that are expressed on these lymphocyte subsets interact with specific chemokines that are selectively expressed in the target tissue. For example, CCR9 is expressed on resident intestinal intraepithelial lymphocytes, lamina propria T cells and on discrete subsets of T cells that traffic to intestinal sites. The ligand for CCR9 is TECK. TECK is expressed in the small intestine, inducing chemotaxis of these cell populations. This suggests that this chemokine/receptor pair contributes to the lymphocyte trafficking in mucosal immune responses.

Fig. 10. Chemokine receptor expression in naive and memory T cells

Similarly, skin-homing CLA+ memory T cells selectively respond to TARC and a novel chemokine CTACK (cutaneous T cell attracting chemokine). TARC is expressed on vessels in the inflamed skin and is thought to bind CCR4 to trigger firm arrest of skin-homing cells and CTACK, which is expressed by the epidermal keratinocytes, may act to attract the T cells out of the vessel and into the dermis and epidermis.

The immune system can construct many specific homing pathways. This is achieved by varying the expression of homing and chemokine receptors and their endothelial and tissue counter-receptors. These interactions occur sequentially in a highly regulated fashion.

IX. EFFECTS OF ACTIVATION ON THE EXPRESSION OF CHEMOKINE RECEPTORS ON T CELLS

As you already know, Th1 cells express IFN-( and are responsible for directing cell-mediated immunity but may also cause disease of the immune system, such as organ-specific autoimmune disease. Th2 cells express IL-4 and IL-5, are important in directing humoral immunity, and are implicated in allergic inflammation. When CD4 T cells differentiate into Th1 versus Th2 cells, they also acquire distinct homing phenotypes. Their homing properties seem to be determined in correlation with their cytokine production programs, and also with their tissue site of antigen exposure.

Figure 12: Overlapping patterns of chemokine receptor, cytokine and skin homing phenotypes in human blood CD4 subsets. The widespread belief that CXCR3 is specific for Th1 and that CCR4 is specific for Th2 cells is clearly misleading. In particular, most CCR4 cells and many CXCR3 cells are ‘non-polarized’, failing to produce either cytokine. Moreover, CCR4 is highly expressed by almost all skin homing CD4 cells in human blood (and skin), and these include both Th1, Th2 and Th0 (IL4+IFNg producing) cells, as illustrated.

This can be taken as an example of the general statement that the homing properties (combination of adhesion and chemoattractant receptors) expressed by any T or B cell seem to correlate both with the cells function in a classical sense (Th1, Th2, B helper CD4 cell; effector vs memory CD8; IgG vs IgA ASC, etc), and with the tissue site where it was generated in response to antigen. It is likely that specialized dendritic cell subsets play a major role in determining the function and homing of antign-activated lymphocytes.

Blood Th1 cells and Th2 cells express overlapping but distinctive sets of chemokine receptors. (Fig. 12).

Fig. 12. Differential expression of chemokine receptors on Th1 versus Th2 effector cells

X. CHEMOKINES DO MORE THAN ATTRACT LEUKOCYTES.

Chemokines and their receptors have now been shown to have additional important functions that extend beyond the regulation of leukocyte migration. CXC chemokines can also influence endothelial migration, and function as angiogenic/angiostatic factors. This has important implications for tumor growth and neovascularization. In smooth muscle cells, chemokines such as IP-10 and IL-8 can modulate proliferation and migration. MCP-1 can induce procoagulant activity that may enhance thrombosis in atherosclerotic plaques. Also, as described above, eotaxin synergizes with IL-5 to rapidly mobilize eosinophils from the bone marrow and also induces myelopoiesis. Targeted disruption of the SDF-1 gene (SDF-1 knockout mice) leads to a severe reduction of myeloid progenitors in bone marrow and absent B cell lymphopoiesis. These findings suggest a key role for SDF-1 in hematopoiesis.

XI. CHEMOKINES AND INFLAMMATION

Early evidence for the role of chemokines in disease was circumstantial, generally based on observations of upregulation of certain receptors or ligands during the disease. For example, as illustrated in Figure 4, in human asthma and models of airway inflammation increased levels of eotaxin and its receptor are observed. Similarly, RANTES and the IFN-(-induced chemokines IP-10 and MIG are elevated during attacks of multiple sclerosis. Their receptors (CCR5 and CXCR3) are present in the inflammatory cell infiltrates. In fact, most infiltrating T cells in diverse inflammatory sites (outside of blood or lymphoid tissues) express both CXCR3 and CCR5.

These data, however exciting, do not formally prove that these chemokines induce the damage. To distinguish causation from mere association, many investigators are turning to immunologically and genetically manipulated mice and mouse models of human disease. There are already some interesting early results suggesting that, indeed, targeting chemokines may allow us to manipulate the immune response. For example, IL-8 has been closely associated with neutrophil-mediated inflammatory disease, e.g. bacterial pneumonia and ischemia-reperfusion injury. Neutralization of IL-8 in rabbits affords almost complete protection from multiple inflammatory challenges, including lung ischemia-reperfusion injury, pleuritis, and glomerulonephritis.

In addition, the CC chemokine MIP-1( has been shown to be present in pathologic specimens from patients with multiple sclerosis. Neutralization of MIP-1( protects against induction of the mouse model of multiple sclerosis, experimental allergic encephalomyelititis (EAE). An even more compelling example is the finding that CCR2-deficient mice have a reduced susceptibility to experimental atherogenesis, in which monocytes (which express CCR2 and respond to the potent CCR2 ligand MCP-1) and macrophages play an important role.

XII. PHARMACOLOGICAL INTERVENTION

Due to their function in vivo, chemokines and their receptors are obvious targets for pharmaceutical intervention. Many commercial labs are searching for small molecules that can disrupt either ligand-receptor interaction or receptor signaling in order to block recruitment, activation or infection of cells. The GPCRs are especially good targets, since there has been prior success in finding antagonists to this family of receptors.

XIV. SUMMARY OF MAJOR POINTS

1. Chemokines are a subset of cytokines that play key roles in homeostasis, inflammation and a host of other pathologies.

2. Certain chemokine/receptor interactions play crucial roles for homeostatic functions, particularly

a) establishing the architecture of the secondary lymphoid organs and for efficiently recruiting naive T cells and antigen bearing (mature) dendritic cells. Thus, chemokines play a key role in efficient initiation of the immune response.

b) The targeted recirculation of memory and effector cells for gut vs skin-associated (allowing more efficient immune surveillance, as well as specialization of immune response modalities such as IgA in mucosal sites, IgG in systemic tissues).

3. Some chemokines are potent inflammatory mediators that attract specific subsets of leukocytes to sites of inflammation.

4. Leukocyte subsets display unique adhesion and chemokine receptor combinations that can be modulated during maturation, differentiation and activation.

5. Specific routes of migration, precise navigation, and orientation within the tissue are determined by combinatorial expression of receptors and sequential encounters with different chemokine gradients. (Acting in combination with adhesion/homing receptors in the multi-step process of recruitment and chemotactic navigation).

6. Chemokine receptor expression is not static. Homing behavior, like other lymphocyte functions, is tightly controlled. Thus, leukocytes undergo constant reprogramming of responsiveness to constitutive and inflammatory chemokines. This reprogramming controls their capacity to selectively migrate to lymphoid, and inflamed tissues.

7. A number of syndromes and diseased tissues are marked by selective expression of certain chemokines that may play key regulatory roles; thus, chemokines and/or chemokine receptor antagonists are likely therapeutic candidates (and many are under current investigation).

Recent References:

1: Kunkel EJ, Butcher EC.

Plasma-cell homing.

Nat Rev Immunol. 2003 Oct;3(10):822-9. Review.

PMID: 14523388 [PubMed - indexed for MEDLINE]

2: Campbell DJ, Kim CH, Butcher EC.

Chemokines in the systemic organization of immunity.

Immunol Rev. 2003 Oct;195:58-71.

PMID: 12969310 [PubMed - in process]

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