Eosinophil Development and Morphology

Similar to neutrophils, eosinophils follow a classic pattern of granulocyte differentiation, passing through blast, promyelocyte, myelocyte, metamyelocyte, and band stages before reaching maturity. Along the way, eosinophils successively acquire morphologically distinct classes of granules. Factors required for eosinophil differentiation include granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3, which also are required for neutrophil differentiation and cannot account for eosinophil commitment. An essential role for IL-5 in eosinophil development has been described, supported by the observation that intravenous administration of IL-5 rapidly results in peripheral eosinophilia. IL-5 may not be completely eosinophil specific, however, because studies in animal models suggest that it is also trophic for B cells. Similar to IL-5, IL-2 can stimulate eosinophilia. The IL-2 effect seems to be mediated through production of IL-5, however. CCL11 (eotaxin-1) also may cause bone marrow release of mature eosinophils and eosinophil precursors via engagement of CCR3 receptors, which are expressed mainly on eosinophils.¹ Cooperation between IL-5 and eotaxins, in particular eotaxin-1, seems to be needed to induce tissue eosinophilia. Knockout mice with targeted deletion of CCR3 show deficiency in gastrointestinal eosinophils. Several transcription factors are involved in the eosinophilic lineage commitment, including the CCAAT/enhancer binding protein family (C/EFB) members, the interferon consensus sequence binding protein (ICSBP), and GATA-1.

When viewed under hematoxylin and eosin staining, eosinophils appear slightly larger than neutrophils (12 to 17 µm). Their nuclei typically are bilobed. Most striking is the presence of large, pink-staining granules. In addition, lipid bodies occasionally may be seen—nonvesicular accumulations of arachidonic acid and other lipids, presumably liberated from plasma membrane. These are not unique, however, and may be detected occasionally in neutrophils as well.

Eosinophils contain at least three distinguishable classes of granules (Table 12-1). Primary granules form first and are analogous to the primary (azurophilic) granules of neutrophils. In contrast to neutrophils, eosinophil primary granules lack myeloperoxidase. Eosinophil primary granules are most numerous in eosinophilic promyelocytes and persist in smaller numbers in mature forms. In mature eosinophils, a lysophospholipase that is present in large quantities (7% to 10% of total eosinophil protein) has been tentatively localized to primary granules, which when released extracellularly precipitates into bipyramidal structures known as Charcot-Leyden crystals. Deposition of these crystals in tissues is taken as evidence of present or past eosinophilia.

Table 12-1 Eosinophil Granule Contents

The large granules visible in mature eosinophils are specific granules that form during the myelocyte stage. When viewed under scanning electron microscopy, specific granules show a dense crystalline core surrounded by an intermediate-density matrix. Because of their large size and number (>90% of overall granule population), eosinophil specific granules have yielded to isolation and immunocytochemical examination, and their contents have been at least partially evaluated. Among the contents probably localized to specific granules are lysosomal enzymes (acid and neutral hydrolases, collagenase, cathepsin, and gelatinase), lectins, and components of the oxidase system. Most distinct is the presence of four highly basic proteins that lend the granule its tinctorial properties. Major basic protein (MBP), an 11,000-kD protein with an isoelectric point value of 11, accounts for more than 50% of the total granule protein and is the major, or possibly sole, component of the crystalline core. Eosinophil cationic protein (ECP), actually a heterogeneous group of several related proteins (18 to 21 kD molecular weight), also is present in large amounts (up to 10% on weight/weight basis).

Eosinophil-derived neurotoxin (EDN) (18 kD molecular weight), the third of the basic granular proteins, is slightly less basic (isoelectric point 8.9) than the aforementioned proteins and is present in smaller quantities. In contrast to MBP and ECP, which have likely roles in host defense, EDN is mainly recognized for its function as a neurotoxin for myelinated neurons, the evolutionary advantage of which is unclear. The Gordon phenomenon, in which injection of eosinophil-laden tissue into an animal produces profound neurologic deficits, is likely due to eosinophil-derived neurotoxin. It has been shown that EDN serves as an endogenous ligand of TLR2, can activate Myd88 in dendritic cells, and shifts adaptive immunity toward a T helper (Th)-2 response, suggesting a pivotal role for esoinophils in the innate-adaptive immune response.² Finally, eosinophil specific granules contain large quantities of eosinophil peroxidase, an enzyme distinctly different from neutrophil myeloperoxidase, but probably subsuming the same function of generating hypohalides for cell killing and activation of latent proteinases. ECP, EDN, and eosinophil peroxidase are localized within the primary granule matrix region. A third population of smaller eosinophilic granules has been identified by virtue of its acid phosphatase and arylsulfatase B content and is present mainly in tissue eosinophils.

Eosinophil Activation and Distribution

Similar to neutrophils, eosinophils undergo activation in response to stimuli and are capable of adhesion, chemotaxis, phagocytosis, degranulation, and O₂⁻ generation. Eosinophils respond to many of the same chemoattractant stimuli as neutrophils, although with different sensitivities. In addition, eosinophils respond to stimuli that do not affect neutrophils, including IL-3, IL-5, regulation upon activation normal T cell expressed and presumably excreted (RANTES), and macrophage inflammatory protein (MIP)-1α. Whether the distribution of these factors is sufficient to explain the tissue distribution of eosinophils relative to other granulocytes is uncertain; however, eosinophils and mast cells secrete IL-3 and IL-5, suggesting their capacity to attract additional eosinophils to sites of atopy. Eosinophils express not only adhesion molecules identified on neutrophils—CD11a/CD18, CD11b/CD18, and L-selectin—but also others, such as the α4β1 integrin VLA-4. IL-4 and IL-13 induce expression of the VLA-4 counterligand, vascular cell adhesion molecule (VCAM)-1, via an eotaxin-1, STAT6-dependent pathway. Oncostatin-M, an IL-6/gp130 family member, also seems to upregulate VCAM-1 in an eotaxin-1, STAT6-dependent manner, and to play a role in eosinophil accumulation in a mouse model.³

Eosinophils also differ from neutrophils in their repertoire of immunoglobulin receptors. Although eosinophils possess immunoglobulin (Ig)G receptors, these are relatively sparse. Instead, the predominant immunoglobulin receptors on the eosinophil surface are high affinity for IgA, consistent with the role of the eosinophil in barrier defense. Although eosinophils are activated by IgG and IgA, they are most potently activated by secretory IgA, probably owing to the presence of a receptor unique for the secretory component. In contrast to earlier teaching, expression of IgE receptors on eosinophils surfaces is minimal and most likely is of little biologic significance.