Chemokines: structure, receptors and functions. A new target for inflammation and asthma therapy?

Five to 10% of the human population have a disorder of the respiratory tract called ‘asthma’. It has been known as a potentially dangerous disease for over 2000 years, as it was already described by Hippocrates and recognized as a disease entity by Egyptian and Hebrew physicians. At the beginning of this decade, there has been a fundamental change in asthma management. The emphasis has shifted from symptom relief with bronchodilator therapies (e.g. β2-agonists) to a much earlier introduction of anti-inflammatory treatment (e.g. corticosteroids). Asthma is now recognized to be a chronic inflammatory disease of the airways, involving various inflammatory cells and their mediators. Although asthma has been the subject of many investigations, the exact role of the different inflammatory cells has not been elucidated completely. Many suggestions have been made and several cells have been implicated in the pathogenesis of asthma, such as the eosinophils, the mast cells, the basophils and the lymphocytes. To date, however, the relative importance of these cells is not completely understood. The cell type predominantly found in the asthmatic lung is the eosinophil and the recruitment of these eosinophils can be seen as a characteristic of asthma. In recent years much attention is given to the role of the newly identified chemokines in asthma pathology. Chemokines are structurally and functionally related 8–10 kDa peptides that are the products of distinct genes clustered on human chromosomes 4 and 17 and can be found at sites of inflammation. They form a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and lymphocytes. The chemokine superfamily can be divided into three subgroups based on overall sequence homology. Although the chemokines have highly conserved amino acid sequences, each of the chemokines binds to and induces the chemotaxis of particular classes of white blood cells. Certain chemokines stimulate the recruitment of multiple cell types including monocytes, lymphocytes, basophils, and eosinophils, which are important cells in asthma. Intervention in this process, by the development of chemokine antagonists, might be the key to new therapy. In this review we present an overview of recent developments in the field of chemokines and their role in inflammations as reported in literature.

FIVE tO 10% of the human population have a disorder of the respiratory tract called 'asthma'. It has been known as a potentially dangerous disease for over 2000 years, as it was already described by Hippocrates and recognized as a disease entity by Egyptian and Hebrew physicians. At the beginning of this decade, there has been a fundamental change in asthma management. The emphasis has shifted from symptom relief with bronchodilator therapies (e.g. 2-agonists) to a much earlier introduction of antiinflammatory treatment (e.g. corticosteroids). Asthma is now recognized to be a chronic inflammatory disease of the airways, involving various inflammatory cells and their mediators. Although asthma has been the subject of many investigations, the exact role of the different inflammatory cells has not been elucidated completely. Many suggestions have been made and several cells have been implicated in the pathogenesis of asthma, such as the eosinophils, the mast cells, the basophils and the lymphocytes. To date, however, the relative importance of these cells is not completely understood. The cell type predominantly found in the asthmatic lung is the eosinophil and the recruitment of these eosinophils can be seen as a characteristic of asthma. In recent years much attention is given to the role of the newly identified chemokines in asthma pathology. Chemokines are structurally and functionally related [8][9][10] kDa peptides that are the products of distinct genes clustered on human chromosomes 4 and 17 and can be found at sites of inflammation. They form a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and lymphocytes. The chemokine superfamily can be divided into three subgroups based on overall sequence homology. Although the chemokines have highly conserved amino acid sequences, each of the chemokines binds to and induces the chemotaxis of particular classes of white blood cells. Certain chemokines stimulate the recruitment of multiple cell types including monocytes, lymphocytes, basophils, and eosinophils, which are important cells in asthma. Intervention in this process, by the development of chemokine antagonists, might be the key to new therapy. In this review we present an overview of recent developments in the field Introduction Diseases characterized by airway inflammation, excessive airway secretion and airway obstruction affect a substantial proportion of the population. These diseases include asthma, (C) 1996 Rapid Science Publishers chronic bronchitis, bronchiectasis and cystic fibrosis. Asthma has been the subject of extensive research for many years. This is not surprising as asthma is a frequently occurring disease with a history of a high morbidity and mortality. Until a few years ago the primary symptoms of mast cells and epithelium cells also contribute asthma were thought to be increased airway to the production of mediators. Some of these responsiveness and recurrent 'reversible' airway mediators, e.g. histamine, LTC4, LTD4 and prosobstruction. This is shown by the definition of taglandin D2 will cause direct contraction of the asthma by the American Thoracic Society dating airway smooth muscle, producing a bronchofrom 1987. spasm and thus an airway obstruction. Various chemotaxins (e.g. LTB4 and chemokines) initiate Asthma is a clinical syndrome characterised by increased responsiveness of the tracheo-bronchial tree to a variety the inflammatory reaction in the airways by of stimuli. The major symptoms of asthma are attacks of attracting leukocytes into the area and hence dyspnea (disorder of breathing), wheezing and cough, preparing for the late-phase reaction.
which may vary from mild and almost undetectable to The second, late-phase occurs in approxisevere and unremitting (status asthmaticus). The primately 50% of the asthmatics (even more in mary physiological manifestation of this hyperresponsiveness is variable airway obstruction. This can take the children) 6 at a variable time after exposure to form of spontaneous fluctuations in the severity of the elicting stimulus. This phase is in essence a obstruction, substantial improvements in the severity of progressing inflammatory reaction and is caused obstruction followingbronchodilatorsor corticosteroids, by the infiltration of, amongst others, airway or increased obstruction caused by drugs or other stimuli obstruction neutrophils and eosinophils. Eosinophils especially play an important role in the The major goal in treatment was to reverse this pathogenesis of asthma. 7 Most of the products airway obstruction, released by these cells have been tested for At the beginning of this decade, a fundamentheir effects on lung tissue. They all have some tal change in asthma management took place, effects (extensively reviewed by Barnes8) but The emphasis has shifted from symptom relief none of them is solely responsible for the with bronchodilator therapies to a much earlier observed phenomenon in the asthmatic reintroduction of anti-inflammatory treatments. 2 action. 9 Based on a growing body of evidence, allergic as well as intrinsic bronchial asthma have been Inflammatory Cells defined as chronic persistent inflammatory disorders. Agreement has been reached that asth-A number of studies have provided information ma can no longer be seen as an equivalent of on cell populations in bronchoalveolar lavage bronchospasm and that the absence of reversi-(BAL) fluid in mild, stable asthmatics with bility of airflow obstruction does not exclude persistent airways hyperresponsiveness and bronchial asthma. asthma. -12 Common findings in these studies, Thus, asthma is now recognized to be a as well as in recent examinations of bronchial chronic inflammatory disease of the airways, mucosal biopsies, 1a4 are the presence of ininvolving amongst others mast cells, eosinophils creased numbers of inflammatory cells, such as and T-lymphocytes. Airway production of cheeosinophils, lymphocytes and mast cells, commokines, cytokines and growth factors in re-pared with normal control subjects with normal sponse to irritants, infectious agents and airway responsiveness. The eosinophils have inflammatory mediators also play an important shown signs of activation, as indicated by inrole in the modulation of acute and chronic creased levels of granular proteins, major basic airway inflammation. 4 Treatment of asthma protein (MBP) and eosinophilic cationic protein should therefore be based on anti-inflammatory (ECP). 15 Both MCP and ECP are cytotoxic for agents rather than bronchodilators. 2 airway epithelium. Azzawi et aL 16

have also
The asthmatic attack can be divided in two demonstrated significant increases in the nummain phases: the immediate-or early-phase ber of activated T-lymphocytes. Mast cells in the asthmatic response and the delayedor lateairways mucosa have exhibited various stages of phase reaction. This division is fairly arbitrary, degranulation, 14 suggesting that mediator rebecause in some subjects only one of the lease is an ongoing process in the airways of phases may be obvious, but it provides a useful stable asthmatics with persistent airway hyperbasis for discussing the physiopathological responsiveness. These inflammatory cells rechanges in the bronchi and the mediators that lease a wide variety of mediators, including are involved. 5 The early-phase, i.e. the initial local release of preformed mediators, newly response, occurs abruptly and is due mainly to synthesized metabolites of arachidonic acid, and spasm of the bronchial smooth muscle. After a soluble pro-inflammatory proteins including ki- 7 challenge with all kinds of stimuli, the alveolar nins and cytokines. Airway epithelial cells macrophages will be activated and produce participate in local cytokine networks and mediators. Other primary effector cells, such as regulate inflammatory airway events by synthe- The non-preformed mediators derived from basophils, eosinophils, mast cells, and other sources are also known as the lipid mediators. Release of inflammatory mediators such as With the exception of platelet activating factor histamine and products of arachidonic acid (PAF), they are products of arachidonic acid metabolism has been demonstrated in BAL fluid metabolism through two different pathways. of patients with asthma. Airway inflammation in The cyclooxygenase pathway is responsible for asthma is a complex series of events triggered the generation of prostaglandins, prostacyclin, by inflammatory stimuli interacting with pri-and thromboxane, while the lipoxygenase pathmary effector cells resident within the airways, way generates leukotrienes and HETES (hydroxy-Release of inflammatory mediators from these eicosatetraenoic acids). In the 5-1ipoxygenase cells may in turn recruit and activate other pathway, arachidonic acid undergoes lipoxyeffector cells or cell-independent systems, with genation to produce leukotriene A4 (LTA4). This generation of other mediators, thus augmenting is subsequently metabolized to LTB4 or LTC4. the inflammatory process. These include pre-LTC4 in turn is rapidly metabolized to LTD4 and formed mediators, such as histamine, mediators LTE4. In physiologic studies the leukotrienes newly synthesized by basophils or mast cells seem to have about the same range of activities after antigen stimulation, such as leukotrienes, as does histamine, and levels of leukotrienes and mediators generated secondarily as a result within the airways are higher in asthma. The of primary mediator release. An example of the cyclooxygenase enzyme catalyzes the incorporalatter is bradykinin, which is generated by the tion of molecular oxygen into the arachidonic action of kallikrein on serum kininogen. Still acid and promotes ring closure to form the other mediators are released from actively relatively unstable cyclic endoperoxides PGG2 recruited cells over longer periods of time (e.g. and PGH2. These are converted to the primary eosinophil granule constituents, cytokines; cheprostaglandins such as PGD2, PGE2, and PGF2. mokines), and their importance in the immuno-Alternatively, the endoperoxides may also be pathogenesis of asthma has been inferred based metabolized to prostacyclin (PGI2) or thromon their detection within the asthmatic airway, boxane A2. Prostaglandin D2 is the predominant or following experimental allergen challenge. 17 prostanoid generated by mast cells; none is Inflammatory mediators may have a variety of generated in human basophils.19 A cyclooxygeneffects on several target cells within the airway ase subtype, cyclooxygenase-2, is induced durand may mimic many of the features found in ing inflammation. Therefore, the prostaglandin asthma. They may lead to contraction of the production will be increased during inflammaairway smooth muscle, either directly or indirtory processes. ectly, through the release of other mediators, or Until recently, PAF was thought to be one of the activation of neural pathway. 8 the most important mediators in the pathogenesis of asthma. This was because it mimics many features of asthma and, in addition to Preformed mediators having physiologic activities much like those of Histamine was the first inflammatory mediator histamine, it also is chemotactic for eosinophils studied, having been synthesized in 1907 and and other inflammatory cells in vivo. 20 Howstudied extensively by Dale and Laidlaw in the ever, it has subsequently been found that other 18 years thereafter. Histamine s generated n mediators such as the leukotrienes may have a basophils and mast cells by the enzymatic similar activity. 17 decarboxylation of histidine. Elevations of hista-Other mediators generated subsequent to mine in BAL fluids have been found in the mast cell and basophil mediator release are the airways of asthmatics, and the levels increase kinins. Bradykinin has effects similar to the strikingly within minutes and even many hours tachykinins (neurokinins A and B, substance P). following antigen challenge. The other pre-When inhaled, it is a potent bronchoconstrictor formed mediators in human basophils and mast and causes a sensation of dyspnoea similar to cells have as yet no well defined roles in the asthma. This is probably due to an action on Mediators of Inflammation Vol 5 1996 :395 sensory nerves within the airways. Levels of kinins have been found to be elevated in asthmatic airways and to increase even further after segmental antigen challenge. 17 Other pro-inflammatory proteins A host of cytokines released by T-lymphocytes and other cells are pivotal in mediating many inflammatory responses in allergic diseases including asthma. Detectable levels of mRNA for TNF, IL-1, IL-3, IL-4, IL-5, and GM-CSF has been reported in biopsies or BAL fluids. A similar but slightly different panel of cytokine proteins has also been observed (e.g. IL-2 and IL-6 have also been detected). The source of these and other cytokines may include not only T-lymphocytes but also macrophages, epithelial cells, mast cells, basophils, and eosinophils. As mentioned above, eosinophilic inflammation is a consistent and prominent finding in asthma. The eosinophil granule proteins (a second category of pro-inflammatory proteins), such as eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and major basic protein (MBP), are highly toxic to epithelium and other pulmonary cells, and inhalation of MBP can induce hyperreactivity. 21 A third category of pro-inflammatory proteins are the chemokines, a special type of cytokines. In the remaining sections of this review the chemokines will be discussed.

Chemokines and Inflammation
Chemokines are structurally and functionally related [8][9][10] kDa peptides that are the products of distinct genes clustered on human chromosomes 4 and 17. They are a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and lymphocytes32 Chemokines are strongly implicated in a wide range of human acute and chronic inflammatory diseases, including arthritis, respiratory diseases, and arteriosclerosis. 3 Additionally, they may play an important role in host defense against infections and in wound healing.
The invasion of the body by pathogenic organisms triggers a cellular response by the immune system that leads to the recruitment of leukocytes. The initial migration of leukocytes toward the site of infection (chemotaxis) is mediated by a variety of molecules, called chemoattractants or chemotaxins. 24 The chemoattractant is the signal that triggers a complex sequence of events dependent on interactions 396 Mediators of Inflammation Vol 5 1996 between adhesion molecules and their complementary ligands on leukocytes. 25 Much of the knowledge concerning leukocyte chemoattractants originates from use of the Boyden chamber which measures chemotaxins in vitro. The invention of the chemotaxis chamber by Boyden in 1962 allowed in vitro quantification of leukocyte movements in defined gradients or soluble chemoattractants. 26 The first chemoattractant for neutrophils demonstrated using this system was the complement fragment C5a. By 1986, the structural and functional properties of the 'classical' chemoattractants N-formyl-methionyl-leucyl-phenylalanine (fMLF), C5a, leukotriene B4, and plateletactivating factor (PAF) had been extensively detailed. 27 Recently, the number of structurally defined chemoattractants for leukocytes has greatly increased, largely due to the identification of the chemokine superfamily. 28 The name 'chemokine' was proposed at the Third International Symposium of Chemotactic Cytokines at Baden in 1992. 'Chemokine' combines the chemoattractant and cytokine properties that have been identified for many of these peptides. Previous to this symposium the chemokines were termed 'intercrines'. Immunologists first detected a member of the intercrine family when Luster et aL 29 in 1985 reported the induction of gene expression for a peptide homologous to platelet proteins in interferon gamma (IFN-y) stimulated macrophages and termed the peptide 'IP-10'. Subsequently, Yoshimura et aL 331 isolated and identified a novel monocyte cell-derived neutrophil chemoattractant, and they were the first to separate this peptide biochemically from IL-1 and TNF, which were previously considered to be responsible for this activity. This novel chemoattractant polypeptide was initially named 'monocyte-derived neutrophil chemotactic factor' (MDNCF). Various investigators have referred to this peptide as a 'neutrophil activating protein' (NAP), MDNCE NAE GCE LCF, LAI and most recently IL-8. 28 The fact that the chemokines have remarkably conserved sequences, distinguishes them from the other chemoattractants and most other cytokines. 27 Although the superfamily is defined by structure, three common functional properties are also apparent. Firstly, chemokines attract one or more myeloid cell types in vitro. Secondly, the production and/or secretion of most chemokines in source cells is induced by pro-inflammatory stimuli such as lipopolysaccharide, tumour necrosis factor-1 (TNF-1) or interleukin-1 (IL-1). Thirdly, all those chemokines that have been tested induce inflammatory infiltrates when injected intradermally into animals, although certain species barriers may exist. 32 The human chemokine polypeptides are 70-90 residues in length and have internal disulphide bonds, comparable with C3a, C4a, and C5a. However, the chemokine and complement fragment sequences are only 15% identical. All chemokines have four cysteine residues which form two disulphide bridges, is Traditionally, the chemokine superfamily has been divided into two subgroups: CXC (c; C is cysteine and X is any amino acid) and CC ([), based on the chromosomal location of the gene, the overall sequence homology and the disposition of the first two of the four conserved cysteine residues ( Fig. 1). All known (z chemokines are 25-90% identical, while all known chemokines are 25-70% identical. Any (z chemokine is 20-30% identical to any chemokine. Recently, the discovery of a new protein suggests that the superfamily may have an additional branch, the 'C' (%,) branch. Lymphotactin, a molecule isolated from pro-T cells, clearly lacks the first and third cysteines in the four cysteine pattern, but shares a large amount of amino acid similarity at its carboxyl terminus with CC chemokines (Fig. 1). 33 The structural analysis, chromosomal location and biological properties of lymphotactin provide strong evidence that this cytokine represents a new class of chemokine. 34,35 Although the chemokines have highly conserved amino acid sequences, each of the chemokines binds to and induces the chemotaxis of a particular class of white blood cells. CXC ((z) chemokines (such as IL-8 and MGSA) stimulate predominantly neutrophils, except for platelet factor 4 (PF-4) and y-interferon inducible protein (yIP-lO). CC () chemokines (such as MIP-I(, MCP-1 and RANTES) on the other hand, do not affect neutrophils but stimulate multiple cell types including monocles, lymphocytes, basophils, and eosinophils. 36 The C (%,) chemokine lymphotactin mainly attracts lymphocytes.
There are probably more structural distinctions to be made, which may explain/enlighten chemokine function. Within the CXC group, the majority of the known proteins contain the amino acid motif Glu-Leu-Arg-Cys-Xaa-Cys (ELRCXC or ELR) at the amino terminal region. These amino acids are absent in certain mem-

Receptor Subtypes
Based on binding specificity and expression in certain cell types, the chemokine receptors can be classified in different ways. According to Schall and Bacon 9 the receptors can, so far, be grouped into four general classes.

Promiscuous receptors
The promiscuous receptor is a receptor that binds chemokines of either CC or CXC classes.
To date, the only example of this receptor is the erythrocyte chemokine receptor (ECKR). Horuk, Chaudhuri, and co-workers have shown that this erythrocyte chemokine binding protein is identical to the Duffy blood-group antigen, which is a receptor for the malarial parasite Plasmodium vivax. 4,41 Shared receptors The shared receptor is a receptor which will bind to more than one chemokine within either the CXC or the CC class. Two examples are the interleukin-8 receptor B (IL-8B receptor) and the CC chemokine receptor-1 (CC CKR-1, also called the MIP-lc/RANTES receptor). The IL-8B receptor binds chemokines with the ELRCXC motif (CXC class), whereas the CC CKR-1 binds several of the CC chemokines.

Specific receptors
These receptors seem to bind only one specific chemokine. The interleukin-8 receptor A (IL-8A receptor) and the monocyte chemoattractant protein (MCP)-I receptor represent this class.

Virally encoded receptors
To date there are two reports of virally encoded receptors. One is encoded by a cytomegalovirus open reading frame, CMV U28, 42'43 and the other from herpes saimiri virus, HS ECRF3. 44 These two receptors are probably shared C-C and C-X-C receptors respectively, that have been transduced by viruses during evolutionay history.
Horuk 24   Additionally, IL-8 is able to desensitize calcium transients elicited by GRO(z and NAP-2, but GROcz and NAP-2 do not desensitize the response to IL-8. 27 The existence of two IL-8 receptors is further supported by the cloning of two cDNAs encoding seven-transmembranedomain receptors, and causing binding 4 of CXC 7 chemokines to cells upon transfection. These products have been referred to as IL-8 receptors A and B, B and A, (z and [3 and Type 1 and Type 2 in literature, but in this review they will be termed A and B following the gene symbols published by Murphy. 27 The deduced sequences of the IL-8A receptor and the IL-8B receptor are highly homologous at the amino acid level (77%), whereas they are 23-30% homologous to other leukocyte chemoattractant receptors (Fig. 3). 27 IL-8A and IL-SB receptors have the highest homology over the membrane-spanning regions, and diverge at the amino and carboxyl termini. 48 The IL-8RA (specific receptor which binds only IL-8) is more widely expressed than the IL-8RB (shared receptor) and is found on neutrophils as well as at low levels on monocytes and monocytic cell lines, melanoma cell lines, T cells, synovial fibroblasts, HL-60 and THP-1 myeloid precursor cell lines. 24 '47 The expression of the IL-8RB receptor is more restricted and Mediators of Inflammation Vol 5 1996 399  24,27 This suggests that the reported ability of IL-8 to attract small numbers of T cells may be mediated by IL-SRA. 27 Regulation of the expression of the IL-8 receptor A/B It has been reported that [125I]IL-8 bound to the IL-SR on neutrophils is rapidly internalized and degraded in lysosomes. [49][50][51] More than 90% of the ligand-bound receptors are endocytosed within 10 min at 37C, and the receptors are recycled, as indicated by their re-expression on the cell surface approximately 10 min later. 49 Inhibitory lysosomotropic agents (agents that show a special affinity for lysosomes), including ammonium chloride, inhibit the internalization process. Ammonium chloride also inhibits chemotaxis, suggesting that chemotaxis may require internalization and reexpression of the IL- of IL-8 required for the down-modulation of IL-8RA was higher than that of IL-SRB. It was found to be impossible to down-modulate IL-8RA/B completely and this is probably due to the two ongoing competitive processes: .downmodulation of receptors by. the agonist and reappearance of receptors after dissociation from the bound ligand. Investigations concerning the recycling of the receptors revealed that after the exogenous IL-8 was removed, the level of IL-SRA continued to increase and reached 85% of the untreated fresh control level during a 1.5-h culture period. In contrast, the level of IL-SRB recovered to only 40% of the control value during a 1-h culture period and then remained at that level. The rapid re-expression of IL-SRA, with respect to IL-SRB, after 'complete' down-modulation supports the hypothesis that IL-8RA may play a more active role in transmitting the IL-8 signal in the inflammatory area compared with IL-8RB. 52 It has been generally accepted that IL-8RA/B have similarly high affinities for IL-8, 225455 although the magnitudes of the affinities reported varied from 0.1 to 4 nM. It is striking, that Chuntharapai and I{im 52 have a different view, by stating that their results obtained from the comparison of the EC50 of IL-8 and the K of each receptor for IL-8 clearly demonstrate that IL-8RB has a higher affinity for IL-8 com-pared with IL-8RA. They detected sevento 13fold and two-to five-fold differences in the EC50 of IL-8 and the Kd values, respectively.
As a result, they proposed a mechanism that could occur during inflammation. In the course of inflammation, resident macrophages and fibroblasts, located at the site of inflammation, secrete IL-8, and this secreted IL-8 gradually reaches nearby blood vessels. At a distant site, the concentration of IL-8 could be in the picomolar range, and at these concentrations IL-8RB would receive the IL-8 signal first and initiate the migration of neutrophils toward the inflammatory area. As neutrophils migrate closer to the site of inflammation, the IL-8 concen-residue was identified. 56 In the GPCR family, the ligand accessible surface is defined as the combination of the extracellular domain and part of the transmembrane domain. It is interesting to note that Asp85, which is located in the second transmembrane domain of the receptor (Fig. 4), is conserved in more than 90% of the members of the GPCR superfamily and may be a key residue maintaining the tertiary structure and proper folding of the receptor.
Replacement of Glu275 or Arg280 from the receptor by Ala causes a complete loss of IL-8 receptor binding. Sequence alignment shows that these residues are strictly conserved in the two human (type A and B), the rabbit, and the mouse IL-8 receptors. This demonstrates that the third extracellular loop of the receptor, which includes these Glu275 and Arg280, is an important functional domain of the receptor.
Although Glu275 appears to be critical for binding, there is no evidence that it is involved in a direct interaction with the Arg6 of IL-8. Hbert and co-workers 56   shown to stimulate chemotaxis and adhesion of Since 1993 new information became available T cells. 8 In addition, the members of this group concerning these receptors and in 1995 cDNAs attracted and activated polymorphonuclear leu-for four human leukocyte CC chemokine recepkocytes (PMN), eosinophils and lymphocytes tots have been cloned. These receptors are with variable selectivity and MIP-I had been designated CC CKR1, CC CKR2A and CC shown to regulate the proliferative capacity of CKR2B (a single gene that produces two splice myeloid progenitor cells 6 variants that differ in their carboxy terminal Competitive inhibition studies using various domains, 63 also known as MCP-1 receptors A CC chemokine ligands demonstrated that these and B) and CC CKR3. The properties of the first chemokines and their receptors exhibited prothree receptors are not fully consistent with miscuity similar to that of the CXC chemokine eosinophil chemotactic responses to CC subfamily and the IL-8 receptors. MCP-1 binding chemokines. 64 MIP-I and RANTES are efffeccould be partially displaced by either MIP-Iz or tive agonists for CC CKR1; however, its RNA is MIP-I. MIP-Iz binding could be completely scarce in eosinophils. Much higher expression displaced by MIP-I, and vice versa, and both is found in neutrophils, monocytes and lymwere partially (30%) displaced by MCP-1. 61 phocytes. 6  between the lungs and tissues. The observation that IL-8 can bind to erythrocytes in a saturable manner, suggested a role for erythrocytes as potential mediators of inflammatory processes.
In contrast to the cloned receptors described, a promiscuous receptor on red blood cells has been characterized, that binds a wide variety of inflammatory peptides of both the CXC and CC groups within the chemokine superfamily. 41 '69 The human erythrocyte chemokine receptor, which was originally postulated to be a 'sink' for IL-87 binds the CXC chemokines IL-8, MGSA and PF-4, and the CC chemokines MCP-1 and RANTES with equal high affinity. 24 Other experiments show that the RBC-bound IL-8 (and most likely other chemokines) does not induce signalling in target cells and that chemokines bound to the red cell surface are inaccessible to their normal target inflammatory cells. 69 Thus, the major role for the red cell chemokine receptor may be one of a clearance receptor for chemoand thus the thermodynamics will strongly tactic and inflammatory peptides in the blood, favour dimerization. 6 Third, IL-8, which con-Due to the broad ligand specificity of the red tains N-methyl-leucine 25, is always monomeric blood cell receptor, it has been designated the and yet remains active. 72 multispecific chemokine (CK) receptor. 1 The fact that the molecular mass of the erythrocyte CK receptor is at least 19 kDa Structure-Activity of CXC smaller than the molecular mass of the cloned Chemokines IL-8 receptors, as well as the ability of the CK The ELR motif receptor to bind to a variety of chemokines, supports the idea that this receptor has a As already mentioned in the introduction of this different structure compared to the cloned IL-8 section the ELR motif is the most critical region receptors. Moreover, the CK receptor showed for interaction with the IL-SR. 36 Mutagenesis no sensitivity to GTP or to GTPyS at concentra-and peptide synthesis showed that out of all of tions which resulted in a 50% reduction in IL- 8 the charged residues, in IL-8 only the aminobinding to plasma membranes prepared from terminal Glu4-Leu5-Arg6 (ELR) sequence was cells transfected with one of the cloned IL-8 absolutely required. The ELR region can be receptors. 7 These data do not support the idea modified such that the receptor binding is that the CK receptor is G-protein linked. It is retained but activity is lost. It is striking that still possible, however, that the erythrocyte CK these antagonists have much lower receptor receptor retains the seven transmembrane doaffinity than IL-8, because usually antagonists main characteristic of this family of receptors, have higher binding affinity than agonists. but that it is uncoupled from its guanine Therefore, this indicates that the ELR motif is nucleotide transducing unit. Alternatively, the both a binding and receptor-activation motif. 73 erythrocyte CK receptor may have a unique Multiple substitutions showed that all three three-dimensional protein structure compared residues of the ELR motif were highly sensitive with that of the cloned IL-8 receptors. Evidence to modification, with the order of sensitivity in support of either of these two possibilities being R >> E > L. 3 Additionally, the ELR conforawaits purification and sequencing of this mation and side chain integrity is critical, as protein. 4 substitution of NMe-Leu and NMe-Arg, or single D-amino acid substitutions greatly reduced activ-Structure-Activity Relationships ity. Adding 'spacer' residues, either Glu or Ala, of Chemokines between the ELR and the cysteine at position 7 resulted in loss of activity with only some The compact, symmetrical nature of the familiar residual binding. ELR effects are subtly context-IL-8 dimer structure led to the widespread dependent since PF-4, but not ,IP-IO or MCP-1, presumption that the dimer form must be binds to IL-8 receptors and activates neutrophils important for function. This notion is extended when its N-terminus is modified to contain further by the finding that MIP-I has an ELR7 4 entirely different mode of dimerization. Thus it ELR-containing N-terminal peptides of IL-8 has been suggested that all the CXC chemo-lack agonist activity, indicating that ELR may be kines have a six-stranded -sheet dimer (three necessary but not sufficient for receptor activaantiparallel -strands from each monomer), tion. The helical C-terminal domain of IL-8 whereas all the CC chemokines an end-on-end also contains determinants for receptor actidimer structure and, moreover, that this strucvation. 27 tural difference may account for the functional differences between the two families. Lusti-Narasimhan et al. 72  GPCRs are mostly small peptides or nonpeptide The residues close to the NH2-terminal end of hormones and mediators. Therefore, it seems the loon i.e. close to cysteine 9, were the most unlikely that the chemokine receptors accomcritical. The major difference between the modate chemokine dimers. Second, protein single substitution and hybrid strategies is that structures are determined at high concentration the hybrids had multiple replacements. Thus, Mediators of Inflammation Vol 5 1996 405 only when several substitutions were made, MCP-1, addition of a residue to the NH2-terminal significant effects were observed. Taken to-or acetylation of the NH2-terminal glutamine gether, the results suggest that the N-terminal resulted in loss of activity. Analogues with the loop comprises a secondary binding site. 27 The NH2-terminal residue converted to Asn, or amino acids 18-22, however, do not appear to residues with nonpolar side chains of varying be essential, as multiple substitutions in this size, had equivalent activity to native MCP-1. region failed to affect activity. Phe21 makes Analogues that had either one, two or three aromatic contacts with Tyrl 3, Phel7, and residues deleted from the NH2-terminal had Trp57 and may have a structural role. Neverlower binding affinity and activity than fulltheless, the possibility that Phe21 has hydrolength native MCP-1. However, MCP-1, 5-76 phobic or aromatic contacts with the receptor had surprisingly significant activity and bound cannot be ruled out. 6 to the MCP-1 receptor. 6 Further deletions resulted in analogues that had significant bind- The disulphides ing to the receptor but no functional activity.
Clark-Lewis and co-workers 36 propose the ex-When the cysteines that form each disulphide istence of an activation region and a receptor bridge were substituted in pairs with the binding region that comprise residues 1-5 and cysteine isostere, a-aminobutyric acid (side 7-10 respectively. Truncation of the NH2-termchain CH2-CH3) both analogues were inactive inal region (up to the first cysteine) of MCP-1 and NMR studies showed significant structural resulted in MCP-1, 11-76, which had residual perturbation, probably due to loss of the binding activity, suggesting that a second region disulphide. Both disulphides are essential for binds, although with low affinity, independently function, indicated by lack of activity of the two of residues 1-10. 75 This contrasts with the CXC analogues. However, they do not seem to be chemokines, where truncation of the ELR motif essential for chemokine function in general, as resulted in absence of receptor binding. Experilymphotactin, which lacks both disulphide ments with hybrids of MCP-1 and MCP-3 led to bridges, is still a chemoattractant. 27 the suggestion that the NH2-terminal is not sufficient to determine activity, and that the NH2-terminal binding site and secondary sites The 30-35 region complement each other to give maximal bind-His33 was analysed extensively due to its intering and activity. The CC chemokines were action with the CXC region and proximity to analysed for the chemotactic activity on monothe two disulphides and the ELR motif, but cytes and THP-1 cells. The order of potency various substitutions had no effect on activity, kines, Clark-Lewis and co-workers 3 hypothesized that there could be similarities in the way ally similar and both stimulate basophils, eosinophils, and lymphocytes, as well as monocytes. This is in contrast to published findings suggesting a distinct mechanism of action for MCP-2. 36 Mutation of Leu25 and Va127 in IL-8

that CXC and CC chemokines interact with
Examination of the sequences of the CXC their receptors. They speculated that the Nchemokines reveals that the highly conserved terminal region would be critical and that leucine, corresponding to Leu25 in IL-8, is in all secondary sites would be necessary. However, cases replaced by a tyrosine in CC chemokines.
instead of just three residues as in the CXC There is also a high degree of conservation chemokines, the entire 10 residues that are among the CXC chemokines of the adjacent NH2-terminal to the first cysteines were impor-Va127 residue, which protrudes from the same IgG immune complexes is mainly neutrophil, whereas mononuclear cells predominate in infections by intracellular pathogens, and in delayed-type hypersensitivity. By contrast, eosinophil and basophil leukocytes are prominent in inflammatory reactions that follow immediate-type allergy, certain parasitic infections and autoimmune events. 78 Moreover, increased numbers of eosinophils have been reported in the lung tissues and airways of patients affected by a number of respiratory pathologies including nasal polyposis and asthma. 39 The in vivo requirements for a trafficking cell are quite complicated, and broadly include four distinct components: circulation, adhesion, diapedesis (migration through junctions between endothelial cells), and migration. 9 Until recently, the mechanisms for the recruitment of a given type of leukocyte into inflamed tissue remained largely a mystery, since most inflammatory cytokines, mediators and chemoattractants have little target cell selectivity. It was suggested that some selectivity may result from the type of adhesion receptors expressed on endothelial cells, e.g. vascular cell adhesion molecule (VCAM) recognition of very late antigen 4 (VLA-4), which is present on monocytes, basophils and eosinophils, but not neutrophils. Furthermore, priming by haematopoietic growth factors can also influence the type of cellular infiltrate. For instance, IL-3 and IL-5 markedly enhance the migration and release responses of eosinophil and basophil leukocytes, but do not affect neutrophils. 78 In the past few years, an improved understanding of cell adhesion and intracellular signalling have helped to unravel some of the details of this important, but complex, process.77 First, leukocytes must overcome haemodynamic forces in order to adhere to the endothelial cell surface, lining the typical vessel wall. Subsequently, they must 'crawl their way' along the endothelial surface, migrate through junctions between endothelial cells (the process of diapedesis), and penetrate the basement membrane before gaining entry into, and migration through, the tissue spaces. 9 The inflammatory process is now thought to be a multi-step phenomenon with contributions from four different families of adhesion molecules, including the selectins and their related carbohydrate and glycoprotein ligands, the integrins and their related immunoglobulin superfamily ligands, and a diverse set of small signalling molecules known as chemokines and their respective receptors. 77 The coordinated expression of adhesion receptors on the surface of the leukocytes and their counterreceptors on the surface of endothelial cells are thought to be a key link in the process. Models of the adhesion component of leukocyte trafficking have been refined into a 'three step' process comprising: (a) rolling of leukocytes along the vasculature (mediated through transient interactions between so-called selectin proteins and their carbohydrate ligands), followed by (b) activation of the cell (induced by classical chemoattractants or chemokines) resulting in firm adhesion (mediated through integrin molecules) leading ultimately to (c) extravasation (crawling along the endothelium, diapedesis, and migration into tissues), presumably in response to a chemoattractant gradient. 9 A key feature is that selectin-carbohydrate, chemoattractant-receptor, and integrin-immunoglobulin family interactions act in sequence, not in parallel. This concept has been confirmed by the observation that inhibition of any one of these steps, with e.g. selectin antagonists, gives essentially complete rather than partial, inhibition of neutrophil and monocyte migration. 79 An important consequence of a sequence of steps, at any one of which are choices of multiple receptors or ligands that have distinct distributions on leukocyte subpopulations or endotheliurn, is that it provides great combinatorial diversity for regu-Mediators of Inflammation Vol 5 1996 407 lating the selectivity of leukocyte localization in lipopolysaccharide or TNF and requires de novo vivo, as has been emphasized in several re-mRNA and protein synthesis. Selectins mediate views.  Each type of leukocyte responds to a function unique to the vasculature, the attacha particular set of area code signals. Inflammament or tethering of flowing leukocytes to the tion alters the expression and location of the vessel wall through labile adhesions that permit signals on vascular endothelium. Chemoattracleukocytes to roll in the direction of the flow. tants provide the greatest number of molecular choices and thus the greatest cellular speci-Carbohydrates and mucin-like ficity'79 molecules The 'three step' process discussed above is oversimplified and refinements to this model Selectins appear to recognize a sialylated carboare required. Firstly, selectins actually mediate hydrate determinant on their counterreceptors. two steps, initial tethering to the vessel wall The carbohydrate ligands for Land P-selectin and rolling, which can be distinguished for Eare O-linked to specific mucin-like molecules.
selectin (see Selectins) by dependence on differ-Mucins are serine-and threonine-rich proteins ent classes of neutrophil ligands. Thus, some that are heavily O-glycosylated and have an selectin-ligand combinations may be important extended structure. in tethering and others in rolling. Leukocytes in the bloodstream travel about 1 000 microns per Chemoattractants second, much too fast for them to sense the chemotactic factor emanating from a site of Chemoattractants are important in activation of damage or infection. The selectins and their integrin adhesiveness and in directing the migracarbohydrate ligands have been found to medtion of leukocytes. In chemotaxis, cells move in iate the initial decelerating event, which is the direction of increasing concentration of a characterized by the tethering and subsequent chemoattractant, which typically is a soluble rolling that allows the leukocyte to test the molecule that can diffuse away from the site of microenvironment adjacent to the inflammatory its production, where its concentration is highsite. 77 est. 84 Leukocytes, which can sense a difference Secondly, the steps are overlapping, rather of 1% in chemokine concentration across their than strictly sequential. Although L-selectin is diameter, move steadily in the direction of the shed from neutrophils soon after activation, chemoattractant. As mentioned earlier, the clasligands for E-selectin remains on the neutrophil sical leukocyte chemoattractant acts broadly, on surface, and thus interactions with E-selectin neutrophils, eosinophils, basophils, and monowill probably persist until transendothelial mb cytes, whereas the chemokines have specificity gration is completed. 79 for leukocyte subsets. 79 This suggests that the chemokines may be centrally involved in specific (transendothelial) migration of leukocyte Selectins subsets. The CC chemokine RANTES is a The selectins or lectin cellular adhesion mole-chemoattractant for memory T cells in vitro cules, include the molecules L-selectin, P-selec-and human MIP-lcz and MIP-1[3 have been found tin and E-selectin. They are transmembrane to be chemoattractant for distinct subpopulamolecules, with a number of extracellular dotions of lymphocytes including naive T-cells and mains homologous to those seen in the comple-B-cells. The CC chemokines MCP-1 and C10 are 9 ment receptors. The extracellular region also thought to induce T cell migration just as has a domain related to the EGF-receptor some of the CXC chemokines e.g. IL-8 and IP-(epidermal growth factor) and a N-terminal 10. The C chemokine, lymphotactin, also shows domain which has lectin-like properties (i.e. it T-lymphocyte chemoattractant activity. Furtherbinds to carbohydrate residues). 8 L-selectin is more, some of the CC chemokines are potent expressed on all circulating leukocytes, except promigratory signals for basophils and eosinofor a subpopulation of memory lymphocytes. Pphils, findings which may be relevant to the selectin is stored preformed in the Weibelunderstanding of allergy and asthma. 9 Palade bodies of endothelial cells and the (z It has long been discussed whether chemoatgranules of platelets. In response to mediators tractants can act in the blood stream, where of acute inflammation, such as thrombin or they would be rapidly diluted and swept downhistamine, P-selectin is rapidly mobilized to the stream by bloodflow. Tethering and rolling of plasma membrane to bind neutrophils and leukocytes through selectins will enhance exmonocytes. E-selectin is induced on vascular posure to chemoattractants by prolonging leuendothelial cells by cytokines such as IL-1, kocyte contact with the vessel wall. However, 408 Mediators of Inflammation Vol 5 1996 retention of chemoattractants at their site of Immunoglobulin superfamily production by noncovalent interactions with members on endothelium as integrin molecules on the vessel wall and within the ligands inflammatory site may also be important. Hepar-Several different immunoglobulin superfamily in binding sites on chemokines provide a mechanism for retention in the extracellular (IgSF) members, expressed on endothelium matrix, enhancement of concentration gradi-bind to integrins expressed on leukocytes.
ICAM-1 (intercellular adhesion molecule 1), ents, and perhaps presentation of chemokines ICAM-2, and ICAM-3 are products of distinct on the endothelium to circulating leukocytes, a5 and homologous genes and were all initially identified by their ability to interact with LFA-1.
Induction of ICAM-1 on endothelium and other Chemoattractant receptors cells by inflammatory cytokines may increase Leukocyte chemoattractant receptors have mul-cell-cell interactions and leukocyte extravasatiple functions. They do not only direct migra-tion at inflammatory sites, whereas constitutive tion, but also activate integrin adhesiveness and expression of ICAM-2 may be important for stimulate degranulation, shape change, actin leukocyte trafficking in uninflamed tissues, as in polymerization, and the respiratory burst7 9 As lymphocyte recirculation. Vascular cell adhesion mentioned earlier, chemoattractant receptors molecule 1 (VCAM-1) is inducible by cytokines are G-protein coupled receptors that span the on endothelial cells and on a more restricted membrane seven times. Neutrophils and lym-subset of nonvascular cells than ICAM-1.79 phocytes express G(zi2 and Gczi3 subunits. The G(x subunits of the tzi class are ADP-ribosylated and irreversible inactivated by pertussis toxin.

The role of chemokines in
All of the biological effects of leukocyte chechemotaxis moattractants are inhibited by pertussis toxin. Coupling through Gai subunits has been confirmed by reconstitution in. transfected cells. 79

Integrins
The selective chemoattractant activities of the chemokines make them ideal candidates to play a key role in the 'sorting' problem of leukocyte trafficking, i.e. getting the correct subpopulation of cells to migrate into the tissues. The chemokines may be even more ideally adapted Integrins are perhaps the most versatile of the to directing leukocyte trafficking, because some several adhesion molecules. They are integral of these proteins can also promote cell subtype membrane proteins that help to bind cells to specific adhesion. 39  In these experiments, MIP-I was immobilized by binding to proteoglycan: a conjugate of heparin with bovine serum albumin (BSA) and cellular proteoglycan CD44 were both effective.

Tanaka et al. propose that MIP-I and other
cytokines with glycosaminoglycan-binding sites will bind to and be presented by endothelial proteoglycans to trigger adhesion selectivity not only of lymphocyte subsets, but also for other cell types. 85 Evidence has now accumulated that chemokines may generally form chemical gradients in an immobilized phase via electrostatic interactions with negatively charged proteoglycans. 39 As a result, it might be convenient to think of chemokines as requiring a scaffolding or presentation molecule in order to interact properly with their related receptor (Fig. 5). This would be an appropriate strategy in vivo, as unless the 410 Mediators of Inflammation Vol 5 1996 chemotactic gradient was preserved in a solid phase, normal conditions of blood flow would wash away any chemoattractant, and a constant replenishment would be required at the source. As the chemoattractant can now be considered as being sequestered and maintained by stable components of the extracellular matrix, a single release of chemokine (as might occur during platelet degranulation) might be sufficient to initiate the inflammatory cascade. The inflammatory response could then be 'fine tuned' as each cell which traffics through a vessel could leave its own signals bound in solid phase.
The model of chemokine involvement in leukocyte trafficking can now be summarized as follows: (a) a chemokine, sequestered in solid phase on the endothelial cell surface, is presented as a signal to trap a specific type of leukocyte as the cell is undergoing selectin-mediated from studies of lymphocyte and neutrophil rolling along the endothelium; TEM. One important difference between eosino-(b) the leukocyte is selectively activated by the phils and neutrophils is that when the CD18 chemokine so that the cell stops rolling and molecule is dysfunctional or absent (as e.g. is becomes firmly adhered; the case in leukocyte adhesion deficiency dis-(c) the adhered leukocyte 'crawls' along the ease) neutrophils do not migrate outside of the chemotactic gradient formed by the chemovasculature into skin and most other tissues; in kines on the endothelium; these patients, eosinophils are still capable of (d) the leukocyte undergoes diapedesis and migrating. Ebisawa et aL 86 speculate that the migrates into the tissue space, while still VLA-4/VCAM-1 system may operate as a failsafe responding to a chemotactic gradient, mechanism for TEM in cases in which the CD18/ICAM-1 pathway is not functional. It is In the following sections we will discuss certain also possible that the VLA-4/VCAM-1 pathway, specific chemokines in relation to asthma and which is operative in monocytes and lympho- Studies using an in vitro model of TEM utilizing as well as in the airways; allergen challenge eosinophils, 6 showed that eosinophil TEM is in causes dramatic increases in levels of these certain ways similar to that of neutrophils. For cytokines. Furthermore, eosinophils of asthexample, activation of endothelial cells with ILmatic subjects display evidence of having been 1 or TNF can significantly increase eosinophil subject to priming in vivo. Although the effect TEM. In contrast, a number of differences of RANTES on BAL eosinophils has not been between eosinophil TEM and that of neutro-assessed directly, one may speculate that the phils or other leukocytes have also been ob-synergy between priming cytokine and RANTES served. Notable among these is the observation chemotaxis would be expected in these BAL that eosinophil active cytokines, including IL-3, eosinophils. It has recently been shown that IL-5, and GM-CSF, can profoundly potentiate the higher levels of RANTES are found in the BAL TEM of eosinophils, while having no effect on fluid of asthmatic individuals than in normal neutrophils. These cytokines are not acting as individuals. Dahinden et aL 87 reported that chemoattractants as they need not be present MCP-3 is also chemotactic for eosinophils. during the TEM assay. Analysis of a host of Although it is approximately one order of chemokine molecules has revealed that espemagnitude less potent than RANTES in activatcially RANTES is an effective eosinophil chemoing eosinophils, MCP-3 must also be considered attractant which has no migration-stimulating as having potential relevance to eosinophilic properties for neutrophils. The chemokines responses in vivo. 87 investigated were IL-8, PF-4, B-TG, yIP-lO, MCAF, MIP-Iz, RANTES, MIP-I and 1-309. In addition, The Effect of IVlCP-3 on injection of human RANTES into dog skin has Eosinophils and Basophils been shown to induce a profound eosinophilic infiltrate. 32 The effect of RANTES was concen-In basophils, MCP-3, MCP-1, RANTES and MIPtration-dependent, was inhibited by antibodies lz all induced cytosolic free-calcium concentraagainst the CD18 adhesion complex on eosino-tion changes and, with different efficacies, phils, and was greatly potentiated by exposure chemotaxis (RANTES from, basophils very effectively. Moreover, but apparently not eosinophils. 6 Due to the MCP-1 is also found in the bronchial epithelium high homology with MCP-3 and the fact that of asthmatic patients. 89 To date the role of MCP-3 and eotaxin are both causing eosinophil MCP-2 has not been elucidated, but as it is not chemotaxis, it was first thought that guinea-pig very potent in chemotaxis and activation, it is eotaxin is the homologue of human MCP-3. thought not to play a critical role in diseases This, however, seems unlikely since eotaxin such as asthma, does not share the chemotactic activity with MCP-3 on other cells than eosinophils. Rothenberg et al. 9 have identified a murine Eotaxin eotaxin, and the structural similarities between Asthma is often characterized by tissue remurine (mouse) and guinea-pig eotaxin indicate cruitment of predominantly eosinophils; che-that both are more closely related to each other mokines acting on eosinophils include certain than to other members of the CC family of CC chemokines, e.g. MCP-2, MCP-3, RANTES chemokines. For example, each protein conand MIP-lc. The CXC chemokine IL-8 is also tains several unique features including a gap in chemoattractive for cytokine-primed eosino-the alignment with the MCPs of two amino phils. However, none of these chemoattractive acids near the N-terminal end of the protein molecules are eosinophil-specific and their rela-and the conservation of basic amino acids near tive importance in selected diseases and experi-the C-terminal end that distinguish it from other mental animal models for allergy remains CC chemokines. It is also noteworthy that the unclear. 9 In contrast to the factors discussed so N-terminal end of MCP-1, including the Nfar, eotaxin, a recently described CC chemo-terminal Gln, which has been shown to be kine, has been proposed as an eosinophil critical for monocyte activity, is replaced by a chemoattractant in a guinea-pig model of aller-His in both routine and guinea-pig eotaxin. 66 91 gic airway inflammation.
Eotaxin appears to These comparisons suggest that eotaxin is a be unique among the chemokines since it distinct cytokine and not a homologue of a causes the selective infiltration of eosinophils known member of the family. only, when injected into the skin and when directly administered to the lungs of naive Eotaxin mRNA expression in different guinea-pigs. In experiments described by Rothenberg et aL 9 migrating cells (induced. by organs eotaxin) were > 95% eosinophils.
As would be expected, eotaxin mRNA is con-In 1993, Griffiths-Johnson and colleagues 91 stitutively expressed in mucosal tissues that reported the purification of a novel chemokine, normally contain eosinophils (skin, lung, and 'eotaxin', from bronchoalveolar lavage (BAL) intestinal tract). 9 Nonetheless, expression of 412 Mediators of Inflammation Vol 5 1996 murine eotaxin is also seen in thymus, lymph Eosinophils contain an armory of chemicals node, and muscle where resident eosinophils necessary for killing parasites. These chemicals are rare. This pattern of mRNA tissue distribu-have been implicated in the damage to airway tion is similar to that seen in guinea-pigs, epithelium that occurs in asthma and may relate although mice have higher expression in the to the observed changes in airway function. thymus and skin and guinea-pigs have higher Rothenberg et al. 92 suggest that eotaxin should expression in the lung. 92 Northern blot analyses be considered as a potentially important endoof total RNA isolated from different guinea-pig genous mediator of eosinophil accumulation in tissue samples revealed easily detectable constivivo. In particular, eotaxin and related moletutive expression of eotaxin in the lung. Lower cules may be involved in both eosinophil levels were detectable in the intestines, sto-accumulation and in chronic structural changes mach, heart, thymus, spleen, liver, testes, and in the asthmatic lung. kidney. In addition, no RNA was detectable in Subsequent to the discovery of guinea pig the brain, bone marrow, or skin. 92 The finding and murine eotaxin, a research team at Leukoof constitutive eotaxin mRNA in mucosal tissues Site (Cambridge, MA) very recently identified where eosinophils are predominantly located human eotaxin, examined its chemotactic activ-(lung and intestines), suggests that eotaxin may ity and characterized its binding to an eosinoplay a role in the normal tissue homing and phil receptor, distinct from the CC chemokine turnover of eosinophils, receptors CC CKR1 (MIP-Iz/RANTES receptor) The unexpected expression of eotaxin mRNA and CC CKR2A,B (MCP-1 receptor). Human in lymphoid tissue and muscle suggests that eotaxin manifested a powerful and selective eotaxin may effect other cell types, because chemotactic activity towards eosinophils in eosinophils are normally not present in these both in vitro and in vivo assays. The fact that tissues, and that eotaxin might therefore have a the chemokines are a 'hot topic' is shown by more widespread function. The expression in the unusual situation that human eotaxin was the thymus and lymph node suggests that already available on the market 9 before its eotaxin may direct lymphocyte homing. 9 identification and functional characteristics had Although the eotaxin gene is expressed at been published. It was only at the beginning of relatively high levels in the lungs of healthy 1996 that the cloning and functional characguinea-pigs without airway inflammation, the terization of human eotaxin was reported by chemotactic activity ascribed to eotaxin has Ponath et al. 94 been reported to be undetectable in the bronchoalveolar fluid of non-antigen-challenged guineapigs. Thus, eotaxin mRNA is constitutively ex-CC Chemokines as a Target for pressed at easily detectable levels in the lung, New Drug Therapy in Asthma when eotaxin activity is still undetectable. After antigen challenge, eotaxin gene expression in To date, no studies concerning strategies antagthe lung is further increased during the early onizing chemokines for asthma therapy are part of the late phase response. Thus, upavailable. The investigations on chemokines so regulation of gene expression, and not constitu-far, have mainly focused on the discovery of tire expression, is associated with the pathogennew chemokines and their receptors, and the esis of airway disease. 92 The up-regulation of understanding of their function. It has been eotaxin mRNA as well as protein after allergen reported that glucocorticoids inhibited the challenge shows that the response is, at least to epithelial expression of RANTES. 95 a large extent, at the level of transcription Glucocorticoids have been used in therapy rather than translation of the existing mRNA, for many years and they are currently the first although the factor responsible for this upchoice treatment for asthmatic patients. These regulation is unknown, steroids however, have many functions e.g. Eotaxin is likely to act in parallel with other inhibition of the production and activity of cytokines generated during the late phase re-many cytokines, reduced generation of eicosponse. For example, IL-5 can prime eosinophils sanoids and PAE reduced cyclooxygenase-2 exto respond to another CC chemokine, RANTES, pression, increased 132 expression, reduced and can promote eosinophil tissue survival and vasodilatation and decreased fluid exudation. As activation. 92 The CC chemokines have also been a result of this wide variety of functions, implicated in wound healing which may be corticosteroids can cause severe side effects e.g.
important in the subepithelial basement memosteoporosis, suppression of endogenous glucobrahe fibrosis that is a prominent feature of the corticoid synthesis, poor wound healing, superasthmatic lung.
infections, tendency to hyperglycaemia and Mediators of Inflammation Vol 5 1996 413 thinning of the skin. 96 These undesired effects can be reduced by local application. In severe asthma however, the steroids are administered systemically. New therapies are the development of drugs that could aim at a selective inhibition of the migration of leukocytes involved in a specific disease. As discussed in this review, chemokines are thought to play a major role in the recruitment of these leukocytes and therefore, drugs that modify the production and/or function of these chemokines might be worth investigating. In asthma the attention should be focused on the chemokines that predominantly cause the recruitment of eosinophils. Modifications are possible at several levels. Firstly, specific antibodies can be developed. For IL-8 there is already an antibody available which selectively blocks the IL-8 function. Antibodies for eotaxin and maybe also for RANTES and MCP-3 may be successful. However, the use of antibodies might in practice not be effective, due to typical pharmaceutical constraints. Secondly, the development of antagonists for the receptors involved, should be considered. There are probably several different chemokines involved, all contributing to some degree. Thus, antagonizing the promiscuous receptors may therefore be the most effective. Preliminary results were obtained by Wells et al. 6 They identified a series of variants of the CC chemokine RANTES that are potent receptor antagonists. These molecules are active in the low nanomolar range, and are able to block CC chemokine effects on purified human cells in vitro. Whether these antagonists will also be able to block CC chemokines effects in vivo remains to be elucidated. Thirdly, the production of chemokines can be inhibited by the use of antisense RNA. In this way translation of the mRNA is prevented and thus the production of the target chemokine. Depending on the homology between the nucleotide sequences of different chemokines, this method might be very selective. From these three options, the development of chemokine antagonists seems the most promising, as they have already been shown to be effective in vitro. Furthermore, their use in practice is not limited due to typical constraints, as is the case for antibodies and peptidic compounds.