I. Introduction and Historical Perspective

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Using a compound microscope and the blood of several animals (including the elephant!), the English anatomist, T. W. Jones, discovered, in 1846, that some white blood cells contained granules that became visible when immersed in hypotonic solutions (Jones, 1846). Although it has been claimed that Jones had discovered the eosinophil (Archer, 1963), it is more likely that he visualized the more abundant neutrophil (Spry, 1988). It was Brown (1898) who probably first detected eosinophils in the blood and bone marrow of patients with eosinophilic leukemia in the latter decades of the 19th century, although the lack of appropriate dyes and staining techniques at that time prevented formal identification. Full credit for the discovery of the "eosinophile" is thus given to Paul Ehrlich (1879) who first noticed that a certain population of white blood cells was stained with a negatively charged, brominated fluorescein compound, eosin, and was so named for that property.

Despite the discovery of eosinophils almost 120 years ago, still relatively little is known of their biochemistry and pharmacology when compared to their highly studied sister cell, the neutrophil. This is perhaps surprising given the critical role of these cells both in host defense (Butterworth and Townley, 1993; Allen and Davis, 1994) and, under certain circumstances, in a variety of diseases, including many, if not all, of those indicated in Table 1. However, a persuasive argument (and one that, through experience, is vigorously championed by the authors of this review!) for the lack of investigation almost certainly reflects the difficulty in obtaining eosinophils in sufficient numbers and of a purity required for detailed studies to be performed and from which unambiguous conclusions can be drawn. Moreover, the process of purification and the effect of previous drug therapy on the ex vivo behavior of human eosinophils invariably leads to alterations in cell function and can make interpretation of results difficult. With the refinement of separation and purification techniques, in particular the use of "negative selection" to remove unwanted leukocytes (Hansel et al., 1989, 1990, 1991b), has come a marked increase in the number of articles published relating to eosinophil biology. Indeed, according to PubMed records, more than 3500 articles have been published since 1990 with a noticeable increase in pharmacological and biochemical content. It thus seems timely to attempt a comprehensive treatise of the pharmacology of the eosinophil, and the authors make no apology for omitting much of the immunology and parasitology which has been elegantly reviewed elsewhere (Capron, 1991, 1992; Weller, 1991; McEwen, 1992; Butterfield and Leiferman, 1993; Butterworth and Thorne, 1993; Wardlaw et al., 1995).

	II. Gross Morphology and Ultrastructure

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Eosinophils are actively motile, terminally differentiated leukocytes derived from the bone marrow, and have been identified in many mammalian and nonmammalian species (Table 2). Human eosinophils are approximately 8 µm in diameter, have a volume of 275 fl and, in addition to their avidity for eosin, exhibit several distinct characteristics that distinguish them from other granulocytes (Sokol et al., 1988; Dvorak, 1991). Generally, normal healthy eosinophils have a bi-lobed nucleus that is filled with partially condensed chromatin (Figs. 1 and 2). In some diseases, however, the number of lobes is increased to more than four (Sokol et al., 1987). A prominent feature of the eosinophil is the presence of many spherical or ovoid granules (Figs. 1 and 2) that occupy approximately one-fifth of the cytoplasm. Four distinct populations of granule (secondary granules, small granules, primary granules, lipid bodies) have been recognized that house a plethora of proteins, many with enzymatic activity (Fig. 2; Table 3). The first morphological marker of the eosinophil is the appearance of granules that are visible at the promyelocyte stage (Zucker Franklin, 1980). Several proteins are found within these structures, including eosinophil peroxidase (EPO),² acid phosphatase, and arylsulphatase B. Despite earlier descriptions to the contrary (Bainton and Farquhar, 1970), these granules are probably precursors of the specific, or secondary, granules that are first seen at the myelocyte stage of maturation (Hardin and Spicer, 1970; Gleich and Loegering, 1984). In eosinophils harvested from humans (Zucker Franklin, 1980; Tavassoli, 1981; Cohen and Ottesen, 1983) and from many other species, including the dog, mouse, rat, goat, guinea pig and rhesus monkey (Jain, 1986), the specific granules feature a prominent crystalloid core containing major basic protein (MBP). Specific granules, containing multiple cores, also have been visualized (Newman et al., 1996) but their occurance is relatively rare (Fig. 3). In addition, other highly charged cationic proteins typified by eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and EPO (Egesten et al., 1986) are located within the noncrystalloid matrix along with a number of cytokines (Fig. 2; Table 3). Differences in the gross morphology of the secondary granules are apparent between species. Thus, in cats, the core is lamellar rather than crystalloid, whereas in cattle, horses, mink, and gorillas the granules lack a central core and appear to be homogeneous when visualized under the electron microscope (Henderson et al., 1983; Jain, 1986; McEwen, 1992).

View larger version (116K): [in this window] [in a new window]   Fig. 1.   Electron micrograph of untreated eosinophils purified from the peritoneal cavity of guinea pigs. A bi-lobed nucleus containing condensed chromatin is shown (large arrow) along with cytoplasm packed with many large, membrane-enclosed, dense crystalloid-containing ovoid granules (smaller arrows). Cells were conventionally fixed (glutaraldehyde/osmium tetroxide). Bar, 1 µm. Original magnification, 18,000×. See II for further details

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Cardinal structures of a human eosinophil. Shown are the typical bi-lobed nucleus (BLN) and the four main granules. The primary (1°) granule is the principle site of Charcot-Leyden crystals, whereas MBP, ECP, EDN, and EPO reside within the classically crystalloid secondary (2°) granule along with a number of cytokines and a host of other proteins many with enzymatic activity. Lipid bodies (LB), which represent a site of lipid mediator biosynthesis, also are found in resting and activated eosinophil where their number is increased along with small granules (SG) that store proteins such as arylsulfase B and acid phosphatase. See II for further details. COX, cyclooxygenase; 5-LO, 5-lipoxygenase; LPLase, lysophospholipase.

View larger version (180K): [in this window] [in a new window]   Fig. 3.   Identification of single and multiple crystalloid cores in specific granules from streptolysin O-permeabilized guinea pig peritoneal eosinophils stimulated with GTPS and Ca2+. Bar, 500 nm. Original magnification, 55,000×. See II for further details.

A population of small granules also has been identified in human tissue eosinophils that are not seen within circulating cells or those in the bone marrow (Parmley and Spicer, 1974). These structures stain intensely for arylsulphatase B and acid phosphatase (Parmley and Spicer, 1974; Dvorak, 1991) and may also contain catalase (Iozzo et al., 1982).

The third type of storage organelle that has secretory properties is the primary granule, which accounts for approximately 5% of all eosinophil granules (Fig. 2). Morphologically, they are roughly spherical, of variable size, and contain no discernible core. In resting eosinophils, primary granules provide the sole location for Charcot-Leyden crystals (Dvorak et al., 1988), which are colorless, and have a characteristic hexagonal, bipyramidal structure with intrinsic lysophospholipase activity (Ackerman et al., 1980; Weller et al., 1980). In activated cells, trace amounts of Charcot-Leyden crystals have been identified within the nucleus and cytoplasm, implying that this protein can be released intracellularly.

The final population of granules is known as lipid bodies and these structures are not membrane-bound (Fig. 2). Approximately five lipid bodies are found per resting eosinophil, although the number can increase when the cell is activated. Lipid bodies are spherical (0.5-2 µm in diameter), electron-dense organelles, and, as the name implies, provide a principle store for arachidonic acid (AA) that is esterified into glycerophospholipids (Weller and Dvorak, 1985; Weller et al., 1991a).

Further description of the morphology of eosinophils in health and disease is beyond the scope of this review, but interested readers should consult articles by Dvorak (1991) and Sokol et al. (1987) which provide a comprehensive treatise of the subject.

	III. Life Cycle, Maturation, and Tissue Distribution

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Eosinophil turnover, or eosinopoeisis, occurs almost exclusively in the bone marrow although ancillary sites of production can include the spleen, thymus, and lymph nodes (Till and McCulloch, 1961; Jain, 1986; McEwen, 1992). The bone marrow from normal individuals contains about 3% eosinophils, of which 37% are mature, nondividing granulocytes, and the remainder are promyelocytes/myelocytes (37%) and metamyelocytes (26%) that exist in "storage" compartments (Spry, 1988, 1993). At any one time, it has been estimated that about 16% of myelocytes are undergoing DNA synthesis (i.e., are in the S phase of the cell cycle), which lasts about 13 h, and that the time taken from the last mitosis until they appear in the blood as mature cells (the emergence time) is approximately 2.5 days (Spry, 1988). The migration of eosinophils from the bone marrow to the blood takes about 3.5 days (Parwaresch et al., 1976). Using [³H]thymidine flash-labeled peripheral blood eosinophils, Walle and Parwaresch (1979) performed studies in three hematologically normal men to estimate the eosinophil reserve capacity in the postmitotic granulocyte compartment in the bone marrow and the effective eosinopoeisis. The results of those experiments demonstrated that mean turnover of eosinophils is approximately 2.2 × 10⁸ cells/kg/day and that the bone marrow provides the largest postmitotic eosinophil reserve capacity (9-14 × 10⁸ cells/kg).

There is compelling evidence that eosinophils are derived from small populations of self-regenerating, hematopoietic stem cells that also are capable of differentiation into the individual lymphomyeloid lineages. The ultimate commitment of stem cells to unipotential progenitors, and their subsequent survival and expansion into mature eosinophils, has been studied extensively, although a complete understanding of the factors and processes by which this occurs still is lacking. It has been suggested that the fate of a hematopoietic stem cell to regenerate or to commit to a multipotential progenitor is purely stochastic (Till et al., 1964; Nakahata et al., 1982; Nakahata and Ogawa, 1982). In the latter scenario, a host of cytokines and other factors are required including interleukin (IL)-6, IL-11, IL-12, granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF; CD117; formerly known as c-kit ligand and Steel factor), and leukemia inhibitory factor (LIF) (Ploemacher et al., 1993; Ogawa, 1994). Further development of multipotential cells into eosinophil progenitors is under the influence of SCF, IL-3, IL-4, granulocyte/macrophage (GM)-CSF, and eotaxin (Kobayashi, 1993, Peled et al., 1998). Interleukin-5 and possibly eotaxin then provide the major driving force for the terminal stages of maturation and release into the blood stream (Clutterbuck et al., 1989; Sanderson, 1993; Palframan et al., 1998a).

In the guinea pig, IL-5 releases eosinophils from the bone marrow by a mechanism that is blocked by the phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitors wortmannin and LY 294002, although the downstream substrates involved in this process are currently undefined (Palframan et al., 1998b). Moreover, the emigration of eosinophils from the marrow precipitated by IL-5 is associated with adhesive interactions involving ₄ and ₂ integrins that act in an opposing manner. In vivo the expression of ₂ integrins is reduced in response to IL-5, whereas the ₄ integrin level remains unchanged. The observation that a ₂ integrin-blocking antibody suppresses IL-5-driven eosinophil mobilization suggests that these adhesion molecules are necessary for effective migration. In contrast, an ₄ integrin-blocking antibody enhances the release of eosinophils from the marrow in response to IL-5, and it has been speculated that this prevents their normal attachment to the bone marrow sinus endothelium (Palframan et al., 1998b). Thus, the egress of mature eosinophils from the marrow involves a number of discrete steps.

Once in the circulation, eosinophils have a half-life of approximately 18 h and a mean blood transit time (26 h) similar to neutrophils (Steinbach et al., 1979). However, the half-life of eosinophils is prolonged when an eosinophilia is precipitated which might be due to an increase in the concentration of certain circulating cytokines that enhance survival (see XII. H) and/or to the saturation of sites through which eosinophils migrate into tissue.

In humans and many domestic animals, eosinophils comprise 2 to 10% of the peripheral leukocytes, but in cows the average titer is approximately 20% (Duncan and Prasse, 1986; McEwen, 1992). The circulating eosinophil count exhibits diurnal variation in some species; thus, in normal human subjects the highest and lowest levels are seen in the evening and the morning, respectively (Horn et al., 1975), whereas the opposite occurs in horses (McEwen, 1992). Eosinophils are predominantly tissue cells and do not reenter the circulation. The gastrointestinal tract, lung, and skin and, in rats, the uterus during dioestrus or oestrogen treatment (see XIV.F) are the principle sites of accumulation (Dembele Duchesne et al., 1991), and histological studies with human tissues have identified columnar epithelial surfaces as particularly rich in eosinophil infiltrates. Large numbers of eosinophils can be found in tissues even when the peripheral blood count is low, which suggests that their longevity is enhanced once they leave the circulation. It has been estimated that the number of eosinophils in the bone marrow and tissues of rats is 300 times higher than the circulating count (Rytomaa, 1960). The tissue distribution of eosinophils in subjects with disease has not been systematically quantified, although it is curious that pathogen-free animals have no eosinophils in their blood and tissue eosinophils are difficult to find. This strongly suggests that an increase in the circulating eosinophil count and retention of eosinophils in tissues is disease-related (Spry, 1993), although this might not apply to the gut (see V.E.1).

	IV. Transcription Factors and Eosinophilia

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Gene transcription is regulated in a highly coordinated and complex fashion by a diverse family of DNA-binding proteins known collectively as transcription factors. In diseases such as those associated with peripheral blood eosinophilia, transcription factors may play a key role in inducing or repressing critical genes that control eosinopoiesis. Perhaps the most universal and ubiquitously distributed transcription factors are activator protein 1 (AP-1) and nuclear factor B (NF-B), which are involved in the regulation and coregulation of many genes. In contrast, other transcription factors have a more cell-specific distribution and regulate the expression of a restricted number of genes. For example, the transcription factors nuclear factor of activated T cells (NFAT) (Rao et al., 1997), guanine-adenine-thymine-adenine (GATA-3) (D. H. Zhang et al., 1997) along with NF-B (Yang et al., 1998) are critically important in controlling the IL-5 and eotaxin genes that are probably essential for the differentiation, maturation, and trafficking of eosinophils (see III.). Similarly, in the lungs of mice that are deficient in the p50 subunit of NF-B, lymphocyte recruitment after allergen provocation is attenuated compared to wild type animals due to a reduction in the secretion of macrophage inflammatory protein (MIP)-1 and MIP-1 (Yang et al., 1998).

Additional genes are also likely to regulate circulating eosinophil number and eosinophilia associated with disease. In individuals with familial eosinophilia, a rare disease encompassed by the generic term hypereosinophilic syndrome that has no allergic or parasitic basis, a locus (or loci) has been identified on region q31-q33 of chromosome 5 which contains the cytokine gene cluster for IL-3, IL-5, and GM-CSF (Rioux et al., 1998). Since no functional polymorphisms were found within the enhancer, promoter, exons, or introns of any of these genes, it has been speculated that a main cause of familial eosinophilia is due to a novel gene that is situated within region q31-q33 (Rioux et al., 1998). Indeed, this idea is supported by the knowledge that greater than 100 anonymous transcripts have been found in that region of human chromosome 5 (Schuler et al., 1996). Martinez et al. (1998) also have identified markers on the same region of chromosome 5 that controls for circulating eosinophil number as a percentage of total white leukocytes.

	V. G Protein-Coupled Receptors and Their Ligands

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G protein-coupled receptors are characterized by an extracellular amino-terminal sequence followed by seven transmembrane-spanning domains, with three extracellular and three intracellular loops, and an intracellular carboxyl terminus. Conserved cysteine residues within the amino-terminal sequence and in the third extracellular loop are thought to form a disulfide bond which is required for ligand binding, while a second disulfide bond is probably formed between conserved cysteine residues within the first and second extracellular loops. The functional responses that result from ligand binding are transduced by G proteins. These are heterotrimeric proteins consisting of , , and  subunits that each exist in multiple isoforms (20 , five , 10 ) in mammalian cells. Several G proteins and/or subunits thereof have been identified in human and guinea pig eosinophils including G_s, G_i3, G₀, G_q/11, and G (Agrawal et al., 1992; Lacy et al., 1995).

In excess of 17 G protein-coupled receptors have thus far been identified on eosinophils (Table 4). These receptors can couple to a vast array of effector proteins that ultimately produce a host of functional responses resulting both in stimulation and suppression of eosinophil activity. These are identified and discussed in detail below.

A. Platelet-Activating Factor

1. Receptors and Signaling. The ether lipid, platelet-activating factor (PAF), evokes its biological effects by interacting with a classical seven transmembrane-spanning receptor that is composed of 342 amino acids and has a molecular mass of approximately 39 kDa (Honda et al., 1991; Nakamura et al., 1991). Radioligand-binding experiments have identified PAF receptors on many cells, including eosinophils. However, until the early 1990s ³H-labeled PAF was the only radioligand available for this purpose and proved to be unsatisfactory for several reasons. Notably, it causes activation of cells and, with prolonged exposure, receptor down-regulation. Furthermore, the lipophilicity of PAF gives rise to high levels of nonspecific binding, "specific" nonreceptor binding, and the labeling of intracellular or internalized receptors, factors that hamper its utility for accurate determination of cell surface receptor density and ligand affinity (Dent et al., 1989). Nevertheless, estimates of K_d (2.3 nM) and B_max (104 fmol/10⁶ cells) have been made for ³H-labeled PAF in human eosinophils (Korth, 1996), and it seems likely that the ligand-labeled sites represent specific receptors because binding was reversed by unlabeled PAF and the PAF antagonist apafant (WEB 2086) (Korth, 1996).

2. In Vitro Effects. PAF is a potent chemoattractant and selectively promotes the migration of eosinophils over neutrophils. The ability of PAF to promote directional migration is significantly increased in eosinophils taken from asthmatic subjects both in remission and during an attack when compared with healthy volunteers (Shindo et al., 1997), suggesting that they have been primed in vivo. Possible candidate-priming agents include GM-CSF, which enhances PAF-induced pulmonary and cutaneous eosinophilia in guinea pigs (Sanjar et al., 1990a) and mice (Yukawa et al., 1992), and IL-3 and IL-5, which prime murine eosinophils for enhanced chemotactic activity induced by PAF (Yukawa et al., 1992). Other proinflammatory effects of PAF include the generation of a plethora of other bioactive lipids (Table 5) and the release of preformed mediators from both the specific and small granules.

3. In Vivo Effects. In guinea pigs, rabbits, and primates, aerosol and systemic administration of PAF results in the extravascular infiltration of eosinophils into the lungs which resembles, both in amplitude and duration, that seen in response to allergen in sensitized animals (Denjean et al., 1983; Arnoux et al., 1988; Lellouch Tubiana et al., 1988; Sanjar et al., 1990b; Gundel et al., 1991; Herd et al., 1992; Wegner et al., 1992). Comparable observations have been made with rats given PAF directly into the pleural cavity (Silva et al., 1989) and in atopic individuals where intradermal administration of PAF produces a cellular infiltrate rich in eosinophils that is reminiscent of the eosinophilia seen in the same subjects after antigen provocation (Henocq and Vargaftig, 1988). Similarly, in individuals with seasonal allergic rhinitis PAF, given intranasally and outside the pollen season, evokes a marked increase in the number of eosinophils (Klementsson and Andersson, 1992) and in the concentration of ECP (Tedeschi et al., 1994) in the nasal lavage fluid.

B. Leukotriene B₄

1. Receptors and Signaling. The BLT, or LTB₄, receptor, which is expressed on guinea pig, mouse, and probably human eosinophils, was cloned in 1997 from retinoic acid-differentiated HL-60 cells. This human receptor is composed of 352 amino acids and is a member of the seven transmembrane-spanning family of G protein-coupled receptors (Yokomizo et al., 1997). A cDNA that encodes a 351-amino acid murine glycoprotein that is 78% identical with the human BLT receptor has also been identified and expressed in Chinese hamster ovary (CHO) cells (Huang et al., 1998). An analysis of [³H]LTB₄ binding to membrane fractions prepared from CHO cells, and retinoic acid-differentiated HL-60 and COS-7 cells transfected with the cDNA for the human and murine LTB₄ receptor show similar binding characteristics, with K_d values of 0.1, 0.14, and 0.15 nM, respectively (Yokomizo et al., 1997; Huang et al., 1998). Binding studies also have identified and partially characterized the BLT receptor on murine and guinea pig eosinophils (Maghni et al., 1991; Ng et al., 1991; Sehmi et al., 1992a; Huang et al., 1998) also using [³H]LTB₄ as the radioligand. However, notable differences are apparent between these studies. Using intact peritoneal eosinophils from guinea pigs, Ng et al. (1991) reported that [³H]LTB₄ interacts with an apparently homogeneous population of binding sites with a B_max of 40,000 sites per cell and a K_d of 2.8 nM, which is approximately 10-fold lower than that reported in the transfection experiments described by Yokomizo et al. (1997). Similar results were reported for the murine receptor (Huang et al., 1998). The sites labeled on eosinophils probably represent functional receptors since various compounds related structurally to LTB₄ compete with the radioligand with affinities that correlate closely with their ability to induce chemotaxis and to evoke the formation of superoxide anions (Ng et al., 1991). Intriguingly, the rank order of potency for the displacement of [³H]LTB₄ from intact peritoneal eosinophils [LTB₄ > 20-hydroxy-LTB₄ > 12R-hydroxyeicosatetranoic acid (HETE) > 12S-HETE > 20-carboxy-LTB₄ > 5S,12S-dihydroxyeicosapentanoic acid (diHEPE) (Ng et al., 1991)] differs from the rank order obtained using membranes from COS-7 cells transfected with the LTB₄ receptor [LTB₄ > 20-hydroxy-LTB₄ > 20-carboxy-LTB₄ > 5S,12S-diHEPE > 12R-HETE > 12S-HETE (Yokomizo et al., 1997)] which might indicate species difference, LTB₄ receptor heterogeneity (see below), and/or the existence of different conformations of a single LTB₄ receptor. With respect to the two latter possibilities, Maghni et al. (1991) reported that [³H]LTB₄ interacts with a heterogeneous population of binding sites on guinea pig alveolar eosinophils; approximately 1000 sites/cell are labeled with high affinity (K_d = 1 nM), whereas 5500 sites/cell are labeled with low affinity (K_d = 63 nM). Identical results have been obtained with guinea pig peritoneal eosinophils (Sehmi et al., 1992a). Thus, a small population (B_max = 900 sites/cell) of receptors for which LTB₄ has high affinity (K_d = 0.3 nM) were identified by radioligand binding along with a large number of sites (60,000/cell) at which LTB₄ has relatively low affinity (K_d = 140 nM). Again, the finding that various metabolites of LTB₄ competed with [³H]LTB₄ for binding to alveolar eosinophils with a rank order of potency in good agreement with their ability to induce chemotaxis (Maghni et al., 1991) supports the belief that the high-affinity sites represent bona fide receptors. Of considerable interest is the role of the two populations of receptor expressed by these cells. Maghni et al. (1991) have considered the hypothesis, posed originally by Goldman and Goetzl (1984), that they mediate different functional responses: the receptor for which LTB₄ has high affinity subserving chemokinesis and chemotaxis, the receptor for which LTB₄ has low-affinity mediating respiratory burst and prostanoid generation. Support for this idea derives from affinity estimates of the LTB₄ antagonist U-75302, which differs significantly (~17-fold) between the two populations of receptor (Maghni et al., 1991). Collectively, the available data suggest that peritoneal eosinophils express the same LTB₄ receptor that is labeled with high affinity by [³H]LTB₄ on guinea pig alveolar eosinophils [albeit at a much higher (~ 40-fold) density]. A reason for the inability of Ng et al. (1991) to identify receptors on guinea pig peritoneal eosinophils for which LTB₄ has low-affinity may relate to the fact that in those studies [³H]LTB₄ was not used at concentrations that would detect the low-affinity sites.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   LTB4-induced signaling in guinea pig peritoneal eosinophils. Scheme A, low concentrations (1 pM to 10 nM) of LTB4 induce a PTX-sensitive increase in the [Ca2+]I and activate the src-related tyrosine kinase lyn and the raf-1/MEK-1/2/ERK-1/2 protein kinase cascade. The increase in [Ca2+]I is due exclusively to influx of extracellular Ca2+, whereas the activation of lyn, is thought to mediate the activation of a Ca2+-dependent PLA2 (possibly cPLA2) and the subsequent release of AA. Scheme B, higher concentrations (100 nM to 1 µM) of LTB4 activate PLC, with attendant generation of Ins(1,4,5)P3, and a Ca2+-independent PLA2 (possibly iPLA2) with a further liberation of AA. LTB4 also is thought to stimulate a tyrosine kinase-dependent pathway that is implicated in the activation of the NADPH oxidase. See V.B for additional details.

2. In Vivo Effects. A number of in vivo animal models have been developed to establish the potential pathogenic role of LTB₄ in allergic eosinophil inflammatory disorders and autoimmune diseases such as multiple sclerosis and asthma. In 1996, Gladue et al. reported that the LTB₄ antagonist CP 105,696 abolished the ability of encephalogenic T lymphocytes, injected into naïve mice, to evoke two cardinal features of experimental allergic encephalomyelitis (multiple sclerosis): paralysis and weight loss. Moreover, the protection was associated with a 97% reduction in the accumulation of eosinophils to the lower spinal cord as determined by light and electron microscopy, and by the level of EPO (Gladue et al., 1996). Those findings have important implications since they show that agonism of LTB₄ receptors results in eosinophil recruitment and that they play a hitherto unrecognized role in the pathogenesis of experimental allergic encephalomyelitis. Clearly, the possible utility of LTB₄ antagonists in the treatment of human multiple sclerosis, and the part eosinophils play in that disease, merits evaluation.

Two receptors (Cys-LT₁ and Cys-LT₂) for the cysteinyl LTs, which include LTC₄, LTD₄, and LTE₄, have been classified pharmacologically but supporting molecular evidence is still awaited. Both receptors couple predominantly through the G_q/11 class of heterotrimeric GTP-binding proteins, and it is highly likely that they are members of the seven transmembrane-spanning family of receptors (see Coleman et al., 1995 for additional details). Currently, selective antagonists are available only for the Cys-LT₁ receptor and these have been used to identify those receptors on eosinophils. However, antagonist affinities have not been calculated and the assignment of eosinophil leukotriene receptors as Cys-LT₁ is equivocal.

1. In Vitro Effects. Relatively little is known of the pharmacological actions of cysteinyl-leukotrienes on eosinophil function compared to those of LTB₄. Although early studies failed to demonstrate that LTD₄ possessed chemoattractant activity (Nagy et al., 1982; Camp et al., 1983), convincing evidence is now available to the contrary. Using a novel in vitro method, which allows the quantification of migration distance and vectorial orientation, it has been shown that LTD₄ is a potent chemoattractant for human eosinophils, with activity in the subnanomolar range. Moreover, LTD₄-induced chemotaxis is antagonized by SK&F 104353, suggesting that Cys-LT₁ receptors are involved (Spada et al., 1994, 1997). In contrast, LTD₄ does not increase the chemokinetic response of eosinophils above spontaneous migratory activity (Spada et al., 1994).

2. In Vivo Effects. In laboratory animals, LTD₄ and LTE₄ given locally and systemmically can stimulate the accumulation of eosinophils into various sites including the skin, eye, and lungs (Spada et al., 1986, 1988; Chan et al., 1990; Foster and Chan, 1991; Woodward et al., 1991; Wegner et al., 1993; Underwood et al., 1996). For example, in one study guinea pig eosinophils were labeled with [¹¹¹In]oxime and injected (i.v.) into recipient animals (naïve and sensitized), and the effect of LTD₄ and allergen on their emigration into the conjunctiva was monitored. Using that model, it was consistently found that LTD₄ and allergen significantly enhanced conjunctival radioactivity by a mechanism that was abolished (LTD₄) and reduced by 50% (allergen) by the Cys-LT₁ receptor antagonist MK-571 (Chan et al., 1990). Significantly, LTD₄ neither promotes the infiltration of eosinophils into the skin of guinea pigs following intradermal administration nor is it active in other ocular anterior segment structures such as the iris, cornea, and ciliary body after topical or intracameral administration (Woodward et al., 1991). Thus, it appears that LTD₄ regulates eosinophilia in a tissue-dependent manner.