The New England Journal of Medicine

Review Articles

Mechanisms of Disease
F R A N K L I N H . E P S T E I N , M . D. , Editor

C HEMOKINES C HEMOTACTIC C YTOKINES T HAT M EDIATE I NFLAMMATION

ANDREW D. LUSTER, M.D., PH.D.

HE attraction of leukocytes to tissues is essential for inflammation and the host response to infection. The process is controlled by chemokines, which are chemotactic cytokines. This review introduces the burgeoning family of cytokines, with special emphasis on their role in the pathophysiology of disease and their potential as targets for therapy.
STRUCTURE AND FUNCTION OF CHEMOKINES

a- and b-chemokines, which contain four cysteines, appear to be the largest families. In the a-chemokines, one amino acid separates the first two cysteine residues (cysteineX amino acidcysteine, or CXC), whereas in the b-chemokines, the first two cysteine residues are adjacent to each other (cysteinecysteine, or CC) (Fig. 1). Two chemokines that do not fit into this classification, lymphotactin,9 with only two cysteines, and fractalkine,10 a membrane-bound glycoprotein in which the first two cysteine residues are separated by three amino acids (CXXXC) and the chemokine domain sits on a mucin-like stalk, may represent additional families. The a-chemokines can be further subdivided into those that contain the sequence glutamic acidleucinearginine near the N terminal (preceding the CXC sequence) and those that do not (Fig. 1).11 The a-chemokines containing the sequence are chemotactic for neutrophils, whereas those not containing the sequence act on lymphocytes. For example, IP-10 and MIG (monokine induced by interferon-g) attract activated T cells,12 and stromal-cellderived factor 1 acts on resting lymphocytes.13
Figure 1. Chemokines and Their Receptors. The chemokines are homologous 8-to-10-kd proteins that are subdivided into families on the basis of the relative position of the cysteine residues in the mature protein. In the a-chemokines, the first two cysteine residues are separated by a single amino acid (CXC), whereas in the b-chemokines, the first two cysteine residues are adjacent to each other (CC). The a-chemokines that contain the sequence glutamic acidleucinearginine preceding the CXC sequence are chemotactic for neutrophils, and those that do not contain this sequence act on lymphocytes. The C chemokine lymphotactin has only two cysteines in the mature protein, and the CXXXC chemokine fractalkine has three amino acids separating the first two cysteines. Chemokine receptors are G proteincoupled proteins that are expressed on subgroups of leukocytes. Four human CXC chemokine receptors (CXCR1 through CXCR4), eight human CC chemokine receptors (CCR1 through CCR8), and one human CXXXC chemokine receptor (CX3CR1) have been identified. MCP denotes monocyte chemoattractant protein, MIP macrophage inflammatory protein, RANTES regulated upon activation normal T-cell expressed and secreted, MDC macrophage-derived chemokine, HCC-1 hemofiltrate CC chemokine, TECK thymus-expressed chemokine, SDF-1 stromal-cellderived factor 1, TARC thymus and activation-regulated chemokine, ELC EBI1 (EpsteinBarr virusinduced gene 1)ligand chemokine, PARC pulmonary and activation-regulated chemokine, SLC secondary lymphoid-tissue chemokine, 6Ckine 6-cysteine chemokine, IP-10 interferon-inducible protein 10, MIG monokine induced by interferon-g, I-TAC interferon-inducible T-cell alpha chemoattractant, DC-CK1 dendritic-cell chemokine 1, LARC liver and activation-regulated chemokine, GCP granulocyte chemotactic protein, GRO growth-regulated oncogene, ENA-78 epithelial-cellderived neutrophil-activating peptide 78, NAP-2 neutrophil-activating peptide 2, and LIX lipopolysaccharide-induced CXC chemokine.

Over 40 chemokines have been identified to date, most of them in the past few years. The relations among chemokines were not initially appreciated, which led to an idiosyncratic nomenclature consisting of many acronyms. When initially identified, these proteins had no known biologic activity but were associated with inflammatory disease (e.g., platelet factor 41 and IP-10 [interferon-inducible protein of 10 kd]2). Only after interleukin-8,3 monocyte chemoattractant protein 1,4,5 and macrophage inflammatory protein 1a and 1b6 had been identified as leukocyte chemotactic factors was it clear that these proteins have in common important structural features and the ability to attract leukocytes. Chemokines are 8-to-10-kd proteins with 20 to 70 percent homology in amino acid sequences. They have been subdivided into families on the basis of the relative position of their cysteine residues.7,8 There are at least four families of chemokines, but only two have been extensively characterized. The

From the Infectious Disease Unit, Partners AIDS Research Center, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School both in Boston. Address reprint requests to Dr. Luster at the Infectious Disease Unit, Massachusetts General Hospital East, Bldg. 149, 13th St., Charlestown, MA 02129. 1998, Massachusetts Medical Society.

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Chemokine
Chemokine receptor

Receptor

Cell Type

MCP-3, -4; MIP-1a; RANTES MCP-3, -4; eotaxin-1, -2; RANTES

CCR1 CCR3

Eosinophil

MCP-1, -2, -3, -4, -5 MCP-3, -4; eotaxin-1, -2; RANTES MCP-3, -4; MIP-1a; RANTES MCP-1, -2, -3, -4, -5 MIP-1a, MIP-1b, RANTES I-309 MDC, HCC-1, TECK Fractalkine SDF-1 MCP-3, -4; MIP-1a; RANTES MCP-1, -2, -3, -4, -5 TARC MIP-1a, MIP-1b, RANTES MIP-3b (ELC) PARC, SLC, 6CKine (Exodus-2) Fractalkine IP-10, MIG, I-TAC

CCR2 CCR3 CCR1 CCR2 CCR5 CCR8 ? CX3CR1 CXCR4 CCR1 CCR2 CCR4 CCR5 CCR7 ? CX3CR1 CXCR3 ? ? CXCR4 CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 ? CXCR4 CXCR1 CXCR2

Basophil

CC

C CC C

Monocyte

Activated T cell

C C

PARC, DC-CK1 Lymphotactin SDF-1

Resting T cell

CXC

C CXC C

MCP-3, -4; MIP-1a; RANTES MCP-1, -2, -3, -4, -5 MCP-3, -4; eotaxin-1, -2; RANTES TARC MIP-1a, MIP-1b, RANTES MIP-3a (LARC, Exodus-1) MDC, TECK SDF-1

Dendritic cell

Glutamic acid leucine arginine

Interleukin-8, GCP-2 Interleukin-8, GCP-2; GRO-a, -b, -g; ENA-78; NAP-2; LIX MCP-1, -2, -3, -4, -5 MIP-1a, MIP-1b, RANTES

Neutrophil

CCR2 CCR5

CXXXC
Chemokine domain

Mucin-like domain

Cytoplasmic domain

Natural killer cell CX3CR1 CXCR3

CXXXC C
Fractalkine IP-10, MIG, I-TAC

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The b-chemokines, in general, do not act on neutrophils but attract monocytes, eosinophils, basophils, and lymphocytes with variable selectivity. Structurally, the b-chemokines can be subdivided into two families (Fig. 1): the monocyte-chemoattractant-proteineotaxin family, containing the five monocyte chemoattractant proteins and eotaxin, which are approximately 65 percent identical to each other, and all other b-chemokines.14 As with the CXC family, the N-terminal amino acids preceding the CC residues of b-chemokines are critical components of the biologic activity and leukocyte selectivity of these chemokines. For example, the addition of a single amino-acid residue at the amino terminal of monocyte chemoattractant protein 1 reduces its biologic activity on monocytes by 100 to 1000 times,15 and the deletion of a single amino acid from that region converts the chemokine from an activator of basophils to an eosinophil chemoattractant.16 Several chemokines undergo N-terminal proteolytic processing after secretion, which alters their activity. For example, the inactive platelet granule chemokine, platelet basic protein, is processed at the N terminal by monocyte proteases to form neutrophil chemoattractant neutrophil-activating peptide 2.17 This may reflect a general mechanism that allows local factors to regulate and amplify chemokine activity.
CHEMOKINE RECEPTORS

Chemokines induce cell migration and activation by binding to specific G-proteincoupled cell-surface receptors on target cells.18,19 Four human CXC chemokine receptors (CXCR1 through CXCR4), eight human CC chemokine receptors (CCR1 through CCR8), and one human CXXXC chemokine receptor (CX3CR1) have been identified (Fig. 1). Chemokine receptors are expressed on different types of leukocytes. Some receptors are restricted to certain cells (e.g., CXCR1 is predominantly restricted to neutrophils), whereas others are more widely expressed (e.g., CCR2 is expressed on monocytes, T cells, natural killer cells, dendritic cells, and basophils). In addition, chemokine receptors are constitutively expressed on some cells, whereas they are inducible on others. CCR1 and CCR2 are constitutively expressed on monocytes but are expressed on lymphocytes only after stimulation by interleukin-2.20 In addition, some constitutive chemokine receptors can be down-regulated; CCR2 is down-regulated by lipopolysaccharide, making the cells unresponsive to monocyte chemoattractant protein 1 (which activates only this receptor), but it remains responsive to macrophage inflammatory protein 1a (which activates CCR1 and CCR5).21 In contrast, the expression of other chemokine receptors is restricted to a cell state of activation and differentiation. For example, CXCR3 is expressed on
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activated T lymphocytes of the T helper type 1 (Th1) phenotype, whereas CCR3, in addition to being expressed on eosinophils and basophils, is preferentially expressed on activated lymphocytes of the T helper type 2 (Th2) phenotype.22 In this way, transient up-regulation of chemokine receptors on leukocytes allows for the selective amplification of either a cell-mediated Th1-type immune response or an allergic Th2-type response. Some chemokine receptors are also expressed on nonhematopoietic cells, including neurons, astrocytes, epithelial cells, and endothelial cells. This suggests that the chemokine system has other roles in addition to leukocyte chemotaxis. Although most chemokine receptors bind more than one chemokine, CC receptors bind only CC chemokines and CXC receptors bind only CXC chemokines. This ligandreceptor restriction may be related to structural differences between CC and CXC chemokines, which have similar primary, secondary, and tertiary structures but different quaternary structures.23 Chemokine receptors, like other members of the family of G-proteincoupled receptors, are functionally linked to phospholipases through G proteins.18,19,24 Many chemokine-induced signaling events are inhibited by Bordetella pertussis toxin, suggesting that chemokine receptors are linked to G proteins of the Gi class. Receptor activation leads to a cascade of cellular activation, including the generation of inositol triphosphate, the release of intracellular calcium, and the activation of protein kinase C.23 Chemokine-receptor signaling also activates small guanosine triphosphatebinding proteins of the Ras and Rho families.25 Rho proteins are involved in cell motility through regulation of actin-dependent processes such as membrane ruffling, pseudopod formation, and assembly of focal adhesion complexes. Thus, chemokine receptors activate multiple intracellular signaling pathways that regulate the intracellular machinery necessary to propel the cell in its chosen direction. Chemokines also interact with two types of nonsignaling molecules. One is the erythrocyte chemokine receptor, called DARC (Duffy antigen receptor for chemokines).26 This receptor, known since the 1950s as the determinant of the Duffy blood group, is expressed on erythrocytes and endothelial cells. Although DARC is structurally related to chemokine receptors, it is distinctive in that both CXC and CC chemokines bind to it and chemokine binding does not induce calcium flux. This receptor may function as a sink for chemokines, clearing them from the circulation. The second type is a group of heparan sulfate proteoglycans. Chemokines are basic proteins, and they bind avidly to negatively charged heparin and heparan sulfate.27,28 Heparan sulfate proteoglycans capture chemokines in the extracellu-

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lar matrix and on the surface of endothelial cells, a process that may serve to establish a local concentration gradient from the point source of chemokine secretion.29
ROLE IN LEUKOCYTE MOVEMENT

Chemokines are thought to provide the directional cues for the movement of leukocytes in development, homeostasis, and inflammation. Leukocyte extravasation from the blood into the tissues is a regulated multistep process involving a series of coordinated interactions between leukocytes and endothelial cells (Fig. 2).30,31 Several families of molecular regulators, such as selectins, integrins, and chemokines, are thought to control different aspects of this process. Selectins facilitate the movement of leukocytes along the surface of endothelial cells (rolling). Chemokines are thought to provide the signals that convert the low-affinity, selectin-mediated interaction into the higher-affinity, integrinmediated interaction that leads to extravasation of leukocytes. Chemokines are believed to control the homeostatic circulation of leukocytes through tissues. The continuous recirculation of lymphocytes through the blood, tissues, and lymphatics in an organized manner brings naive lymphocytes into the lymph nodes, where they encounter antigen and are transformed in memory lymphocytes that migrate into inflamed tissue to ensure immunity. Macrophages, eosinophils, and mast cells also migrate into tissue. Although these cells are produced in the bone marrow, they reside primarily in other tissues. The role of chemokines in regulating the movement of cells into tissues has begun to be elucidated on the basis of studies in mice deficient in a particular chemokine. For example, stromal-cellderived factor 1 is critical for the migration of myeloid precursors from the fetal liver to the bone marrow,32 and eotaxin is important in the recruitment of eosinophils into tissues.33 The dramatic increase in the secretion of chemokines during inflammation results in the selective recruitment of leukocytes into inflamed tissue. Chemokines have been detected during inflammation in most organs, including the skin, brain, joints, meninges, lungs, blood vessels, kidneys, and gastrointestinal tract. They have also been identified in many types of cells during inflammation in these organs, suggesting that most, if not all, cells can secrete chemokines, given the appropriate stimulus. The main stimuli for chemokine production are early proinflammatory cytokines, such as interleukin-1 and tumor necrosis factor a, bacterial products, such as lipopolysaccharide, and viral infection.7 In addition, interferon-g and interleukin-4, products of Th1 and Th2 lymphocytes, respectively, can induce the production of chemokines and also synergize with in-

terleukin-1 and tumor necrosis factor a to stimulate chemokine secretion.34,35 If proinflammatory cytokines stimulate secretion of many of the same chemokines, how can an inflammatory response be specific? In asthma, for example, eosinophils accumulate in the airways, whereas the host response to bacterial pneumonia is dominated by neutrophils. As pointed out above, the recruitment of leukocytes into tissues is a multistep process in which chemokines participate but do not act alone. Chemokines often act in concert with other cytokines to cause tissue infiltration, by increasing the circulating pool of a given leukocyte and up-regulating particular adhesion molecules, as well as increasing leukocyte responsiveness to a chemokine. For example, eotaxin and interleukin-5 together cause tissue eosinophilia,36 and interleukin-5 and interleukin-3 prime basophils to release histamine and leukotriene after stimulation by monocyte chemoattractant protein.37
ROLE IN INFLAMMATORY DISEASES

The secretion of chemokines has been detected in a wide variety of diseases (Fig. 3).7 It is likely that in these diseases chemokines cause the accumulation and activation of leukocytes in tissues. The capacity to control precisely the movement of inflammatory cells suggests that the various chemokines and their receptors might provide novel targets for therapeutic interventions. The type of inflammatory infiltrate that characterizes a specific disease is controlled, in part, by the subgroup of chemokines expressed in the diseased tissue (Fig. 3). For example, in many acute disease processes, such as bacterial pneumonia and the acute respiratory distress syndrome, there is a massive influx of neutrophils into the tissue. The concentration of potent neutrophil chemoattractants, such as interleukin-8, is increased in bronchoalveolar fluid from patients with these pulmonary diseases.38 In viral meningitis monocytes and lymphocytes are recruited into the tissue, concentrations of chemokines that are active on these cells, such as IP-10 and monocyte chemoattractant protein 1, are increased in cerebrospinal fluid, and the increases are correlated with the extent of mononuclear-cell infiltration of the meninges.39 Tissue infiltration by lymphocytes and macrophages occurs in many chronic diseases. Activated lymphocytes accumulate in the granulomatous lesions that are characteristic of tuberculoid leprosy and sarcoidosis, and high concentrations of IP-10 have been detected.40 Moreover, the concentration of IP-10 in bronchoalveolar fluid from patients with active sarcoidosis is correlated with the number of activated T lymphocytes in the fluid (unpublished observations). In atherosclerosis, macrophages and lymphocytes are the chief inflammatory cells found
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Red cells

Lumen

DARC

Integrin

Adherence
Activated integrin

Endothelium

Activation

Rolling Extravasation
Intercellular adhesion molecule Mucin Cell-surface heparan sulfate proteoglycans Selectin

Inflammatory trigger (e.g., infection, allergen, autoantigen, alloantigen, tumor) Activated recruited leukocyte

Chemokine receptor

Interleukin-1 receptor or tumor necrosis factor receptor

Matrix heparan sulfate proteoglycans Chemokine

Leukocyte

Interleukin-1 and tumor necrosis factor

Figure 2. Chemokine Regulation of Leukocyte Movement. Chemokines are secreted at sites of inflammation and infection by resident tissue cells, resident and recruited leukocytes, and cytokine-activated endothelial cells. Chemokines are locally retained on matrix and cell-surface heparan sulfate proteoglycans, establishing a chemokine concentration gradient surrounding the inflammatory stimulus, as well as on the surface of the overlying endothelium. Leukocytes rolling on the endothelium in a selectin-mediated process are brought into contact with chemokines retained on cell-surface heparan sulfate proteoglycans. Chemokine signaling activates leukocyte integrins, leading to firm adherence and extravasation. The recruited leukocytes are activated by local proinflammatory cytokines and may become desensitized to further chemokine signaling because of high local concentrations of chemokines. The Duffy antigen receptor for chemokines (DARC), a nonsignaling erythrocyte chemokine receptor, functions as a sink, removing chemokines from the circulation and thus helping to maintain a tissuebloodstream chemokine gradient.

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Inflammatory Disease
Acute respiratory distress syndrome

Infiltrate
Neutrophil

Chemokine
Interleukin-8; GRO-a, -b, -g; ENA-78 MCP-1, -4; MIP-1a; eotaxin; RANTES

Figure 3. Role of Chemokines in Various Inflammatory Diseases. Inflammatory diseases are characterized by the selective accumulation of leukocyte subgroups, a process controlled by the expression of certain chemokines. Each disease has a characteristic inflammatory infiltrate in which chemokine messenger RNA or protein concentrations have been shown to be up-regulated. The chemokine abbreviations are explained in the legend to Figure 1.

Asthma

Eosinophil, T cell, monocyte, basophil

Bacterial pneumonia

Neutrophil

Interleukin-8, ENA-78

Sarcoidosis

T cell, monocyte

IP-10

Glomerulonephritis

Monocyte, T cell, neutrophil

MCP-1, RANTES, IP-10

Rheumatoid arthritis Monocyte, neutrophil Osteoarthritis

MIP-1a, MCP-1, interleukin-8, ENA-78 MIP-1b

T cell, monocyte Atherosclerosis Monocyte, neutrophil, T cell, eosinophil

MCP-1, -4; IP-10

Inflammatory bowel disease

MCP-1, MIP-1a, eotaxin, IP-10, interleukin-8

T cell, neutrophil

Psoriasis Neutrophil, monocyte

MCP-1, IP-10, MIG, GRO-b, interleukin-8 Interleukin-8; GRO-a; MCP-1; MIP-1a, -1b MCP-1, IP-10

Bacterial meningitis

Viral meningitis

T cell, monocyte

in the diseased blood vessels. As progenitors of lipidladen foam cells and a source of growth factors that mediate intimal hyperplasia, these inflammatory cells may be central to the pathogenesis of atherosclerosis. Although the mechanism of monocyte recruitment in atherosclerotic lesions is unknown, monocyte chemoattractant protein 1 has been detected in diseased carotid arteries but not in normal carotid arteries.41 In asthma, rhinitis, and atopic dermatitis, there is selective accumulation and activation of eosinophils and mast cells, and mediators derived from these cells participate in the pathogenesis of these allergic diseases.42 Agents that induce the release of histamine from mast cells and basophils, so-called histamine-releasing factors, play an important part. Chemokines, especially eotaxin and the monocyte chemoattractant proteins, are potent eosinophil chemoattractants and histamine-releasing factors, making them particularly important in allergic inflammation.14 In fact, these chemokines may be the main histamine-releasing factors in the absence of antigen and IgE antibody. Many chemokines have been detected in the airways of patients with asthma.43-45 In addition, several chemokines that act on eosinophils are increased in the epithelial tissue in patients with atopic dermatitis, allergic rhinitis, or asthma after an antigen challenge, making it likely that these chemokines are a molecular link between antigen-specific immune activation and the migration of eosinophils into tissues.34,46,47 Ulcerative colitis and Crohns disease are characterized by chronic inflammation with superimposed acute inflammatory exacerbations. In the chronic phase, macrophages and lymphocytes infiltrate the bowel, and in the acute phase, neutrophils and perhaps eosinophils leave the circulation and enter the intestinal mucosa. Many chemokines are markedly increased in intestinal tissue from patients with ulcerative colitis or Crohns disease.35,48,49 In psoriasis, the lesions contain neutrophils and activated T cells, and the neutrophil chemoattractants interleukin-8 and GRO-a (growth-related oncogene a, although it is not an oncogene). The activated T-cell chemoattractants IP-10 and monocyte chemoattractant protein 1 are present in psoriatic plaques but not in normal skin.40,50 In addition, successful treatment of

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psoriatic plaques results in decreased IP-10 in diseased skin.40

ROLE IN INFECTIOUS DISEASES

another intriguing link between the chemokine system and diseases in humans.
MODULATION OF ANGIOGENESIS, TUMOR GROWTH, AND STEM-CELL PROLIFERATION

Pathogenic organisms bind to receptors on leukocytes to gain access to the cytoplasm and nucleus of cells (e.g., EpsteinBarr virus binds to complement receptor 3, and rhinoviruses bind to intercellular adhesion molecule 1). The chemokine receptors serve as coreceptors for two important human pathogens, plasmodium and the human immunodeficiency virus (HIV). Plasmodium vivax binds to the DARC receptor on erythrocytes,26 and HIV binds to several chemokine receptors. By facilitating entry into cells, these receptors determine viral tropism.51-57 CXCR4 is a coreceptor for strains of HIV type 1 (HIV-1) that infect T-cell lines (T-tropic strains), and CCR5 is a coreceptor for HIV-1 isolates that infect macrophages and activated T cells (M-tropic strains) (Fig. 4). RANTES (regulated upon activation normal T-cell expressed and secreted) and macrophage inflammatory proteins 1a and 1b, which are CCR5 ligands, and stromal-cellderived factor 1, a CXCR4 ligand, block the entry of M-tropic and T-tropic HIV, respectively, into cells.58-60 The importance of chemokine receptors in the pathophysiology of HIV infection became apparent when it was discovered that a polymorphic variant of CCR5 could explain the observation that certain persons who are at high risk for HIV-1 infection remain uninfected.61-63 In persons who are homozygous for a 32-base-pair deletion in the gene for CCR5, a functional CCR5 protein cannot be synthesized, and such persons are not found in HIV-1 positive cohorts. In addition, cells from persons with this mutation cannot be infected with HIV-1 in vitro.64 Furthermore, in persons who are heterozygous for the mutation, the rate of progression of HIV-1 infection is slower than in those without the mutation.61 Although the molecular mechanism of the fusion event is not known, HIV glycoprotein 120, CD4, and a chemokine receptor associate on the cell membrane before HIV enters the cells.65-67 Pathogenic organisms express cytokines and cytokine receptors that are important in infection (e.g., poxviruses encode functional interleukin-1b and interferon-g receptors). Many of the herpesviruses express chemokine-receptor homologues, and many of these homologues can bind chemokines. Kaposis sarcomaassociated herpesvirus 8 was recently shown to encode a constitutively (agonist-independent) active chemokine receptor that stimulated cellular proliferation.68 In addition, human herpesvirus 6, Kaposis sarcomaassociated herpesvirus 8, and the molluscum contagiosum virus encode CC chemokine homologues. Although the role of these viral chemokine and chemokine-receptor homologues in the infectious process is unknown, they represent
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Chemokines can also modulate angiogenesis and tumor growth and inhibit stem-cell proliferation. Platelet factor 469 and IP-1028,70 inhibit neovascularization, tumor growth, and metastasis.71 In contrast, interleukin-8 promotes angiogenesis and tumor metastasis.71,72 The mechanism underlying the inhibition of angiogenesis may be related to the ability of certain chemokines to displace other growth factors, such as basic fibroblast growth factor and transforming growth factor a, from heparan sulfate sites on endothelial cells. The ability of certain chemokines to inhibit stemcell proliferation may have therapeutic applications.73 Macrophage inflammatory protein 1a and analogues with antistem-cell but not proinflammatory activity are being developed as adjuncts to cancer chemotherapy. If they can selectively arrest stem-cell replication, then high-dose chemotherapy may be better tolerated because of decreased stem-cell toxicity. Monocyte chemoattractant proteins 1, 2, and 3 have been isolated from glioma and osteosarcoma cells.4,74 Many other chemokines are produced by tumor cells in vitro and are present in human tumors.34,35,71 However, the overall role of chemokines in tumor biology is unclear. Tumor-associated leukocytes may stimulate or inhibit tumor growth. They can promote growth by supplying growth factors and promoting angiogenesis and can inhibit growth by enhancing the host response to the tumor.
STUDIES IN ANIMALS

Animal models of disease have been useful in studying the relation between chemokine expression and pathophysiologic processes.75 In certain inflammatory responses, a single chemokine plays a major part in the recruitment of a subgroup of leukocytes. For example, there are numerous chemokines that act on neutrophils, but a neutralizing antibody against interleukin-8 completely blocks reperfusion-associated lung injury in rabbits.76 In addition, even though other b-chemokines have many of the actions of macrophage inflammatory protein 1a, including an ability to bind to the same receptor, in studies of mice with a deletion of the gene for this chemokine, myocarditis did not develop when the mice were infected with coxsackievirus B3, and there was a significant reduction in the histologic degree of pneumonitis after infection with influenzavirus.77 The recruitment of leukocytes in other inflammatory responses depends on multiple chemokines. In animals with allergic pulmonary inflammation, the expression of eotaxin, macrophage inflammatory

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M-tropic HIV

M-tropic HIV

T-tropic HIV

T-tropic HIV

gp120 V3 loop V3 loop

MIP-1a, MIP-1b, or RANTES

gp120 V3 loop V3 loop SDF-1

CD4 Cell membrane

CCR5

CD4

CCR5

CD4

CXCR4

CD4

CXCR4

Entry of M-tropic HIV strains

Entry of M-tropic HIV strains blocked

Entry of T-tropic HIV strains

Entry of T-tropic HIV strains blocked

Figure 4. Chemokine Receptors as Obligate Coreceptors for HIV Entry into Cells and Chemokine Inhibition of HIV Entry. HIV glycoprotein 120 (gp120) binds to CD4, resulting in a conformational change that exposes the V3 loop in gp120 and permits subsequent interaction with a chemokine receptor. To gain entry into cells, macrophage-tropic (M-tropic) HIV-1 uses CCR5 predominantly, and the T-celltropic (T-tropic) HIV-1 uses CXCR4 predominantly. Macrophage inflammatory proteins (MIP) 1a and 1b and the RANTES (regulated upon activation normal T-cell expressed and secreted) chemokine, ligands for CCR5, block M-tropic HIV-1 from entering cells. Stromal-cellderived factor 1 (SDF-1), a ligand for CXCR4, blocks T-tropic HIV-1 from entering cells.

protein 1a, and monocyte chemoattractant proteins 1, 3, and 5 precedes the massive airway recruitment of mononuclear cells and eosinophils.14,78-80 On the basis of studies using antibodies that inhibit the action of these chemokines and studies of mice with a targeted disruption of the eotaxin gene, it is clear that all these chemokines participate in the recruitment of eosinophils into the airways.33,80-82 Other studies in animals suggest that although a given chemokine may be only partly responsible for the recruitment of a subgroup of leukocytes, it may be largely responsible for certain pathophysiologic aspects of a disease. For example, in animals with acute immune-complexmediated glomerulonephritis, the glomerular lesions contain neutrophils, and there is extensive fusion of the glomerular foot processes, with increased urinary protein excretion. The administration of an antiinterleukin-8 antibody reduces neutrophil infiltration by only 40 percent but completely prevents the fusion of epithelial foot processes and urinary protein excretion.83

Certain chemokines have a critical role in development. Mice deficient in stromal-cellderived factor 1 die perinatally, with defects in B-cell lymphopoiesis and the recruitment of hematopoietic progenitors from the fetal liver into the bone marrow, as well as a ventricular septal defect.32 This dramatic phenotype underscores the possibility that aside from directing the migration of leukocytes to sites of inflammation and infection, chemokines may play an important part in orchestrating the movement of cells during development.
CONCLUSIONS

Chemokines appear to have the capacity to control precisely the movement of leukocytes. The roles of chemokines in the pathophysiology of disease are still being defined, but there is growing evidence from studies in animals that the neutralization of chemokine activity may have therapeutic value. Chemokine or chemokine-receptor antagonists may inhibit autoimmune, allergic, and septic processes. In
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addition, chemokines may augment the host response to infection, tumors, and vaccines. Chemokines in combination with other cytokines may provide more effective antitumor therapy than either alone, as shown in a study of lymphotactin combined with interleukin-2.84 Finally, chemokines or their analogues may be clinically useful as inhibitors of HIV-1 infection and disease progression. The chemokines are a fascinating family of cytokines that we are only beginning to understand. It is likely that additional chemokines and chemokine receptors will soon be discovered. The challenge for the future will be to understand the role of chemokines in the pathophysiology of disease.
Supported by grants from the National Institutes of Health (R01CA69212 and R01-AI40618). Dr. Luster is the recipient of a Cancer Research Institute Benjamin Jacobson Family Investigator Award and a Culpeper Medical Scholars Award.

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