Eosinophils in Health and Disease

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Chapter | 1 Historical Overview and Perspective on the Role of the Eosinophil in Health and Disease 3

successful clinical trials of arsphenamine for the treatment this simple air-drying procedure was fortunate because the
of syphilis in 1910. He died in 1915 at the age of 61 of integrity of neutrophils and eosinophils is destroyed by the
diabetes with cardiovascular and renal disease. With his chemical fixation procedures used at that time.3 Ehrlich
death, the early period of eosinophil research ended. distinguished among the granules of leukocytes, and he
Ehrlich had the good fortune to be the beneficiary of referred to them as alpha and beta granules. Alpha granules
a remarkable blossoming of chemistry in Northern Euro- bound acid coal tar dyes, especially eosin. In 1879, in
pean countries such as England, France, and Germany. In a publication, Ehrlich referred to the cells containing alpha
1824, Justus von Liebig had established the world’s first granules as eosinophils.6 Ehrlich’s work was seminal in
major school of chemistry and taught a generation of that, in addition to discovering eosinophils, neutrophils,
chemists, including names that have become bywords for basophils, lymphocytes, and mast cells, he also established
chemistry, such as August Kekulé, Emil Erlenmeyer, and the methods for counting blood cells so that quantitative
August Wilhelm von Hofmann. Von Hofmann determined associations with disease states could be made.7 While
the nature of aniline and laid the scientific basis for the Thomas Wharton Jones likely first identified eosinophils by
dyestuff industry. In 1865, Friedrich Engelhorn established virtue of their refractile granules,8 his observations did not
the Badische Anilin- und Soda-Fabrik (Baden Aniline and provide the tools for the ready identification and quantifi-
Soda Factory; BASF), and that company came to exemplify cation provided by Ehrlich.
a special symbiosis between business and scientific Histology of the Blood, written by Ehrlich and Adolf
research.4 Heinrich Caro was trained in the laboratory of Lazarus, is presently available in eBook format from the
Robert Bunsen and joined BASF in 1868. He is regarded as Project Gutenberg website.7 In it, Ehrlich and Lazarus
being responsible for the company’s successes in the dye describe the methods for staining blood cells, the need for
industry and developed methylene blue. He and his meticulously produced glass coverslips, the dyes employed
colleagues built the company from a small enterprise to an and the care for their use and, in the case of the eosinophil,
international giant on the basis of the synthesis of chemical the diseases associated with increased numbers of blood
dyes. In 1874, he synthesized eosin by the reaction of eosinophils. For example, counting eosinophils per volume
fluorescein with bromine in glacial acetic acid. Evidently, of blood provided us with the normal numbers of blood
Caro knew Greek mythology because he named the dye, eosinophils in healthy subjects vs. increased numbers of
eosin, by reference to Eos, the Titan goddess of the dawn. eosinophils in asthma,9 pemphigus (one wonders if the
Eos is described by Nonnus of Panopolis as opening the patient more likely suffered from bullous pemphigoid),10
gates of heaven with rosy fingers and golden arms so that urticaria,11 helminthiasis,12 and in numerous other clinical
Helios, her brother, could ride his chariot across the sky situations, including postfebrile periods (for example, after
every day.5 an acute attack of malaria), in malignant tumors, after
Ehrlich transferred the use of dyes from staining bio- splenectomy, and after medications. Moreover, Ehrlich was
logical fabrics, such as wool, silk, and cotton, to staining aware of the association between CharcoteLeyden crystals
cells and tissues. His special genius was the recognition in feces and eosinophilia, especially in patients with
that dyes could make chemical distinctions, and, from the helminthic infection. He found that eosinophils develop in
earliest days of his career, he employed them to investigate the bone marrow before migrating to the blood, and that
cells in blood and tissues. He discovered that cells stained they are a distinct cell line, such that a transition between
differentially and that he could distinguish among them by neutrophils and eosinophils is not observed.
their staining properties. His doctoral thesis, submitted to Ehrlich suggested that eosinophils and neutrophils
the University of Leipzig in 1878, dealt with the chemical possessed different chemotactic irritability and that eosin-
composition and classification of aniline dyes and their use ophils only migrate to sites where a specific stimulating
in general histology. The thesis included a section on substance is present; here, he anticipated the discovery of
granulated connective tissue cells for which he proposed chemokines, including eotaxins. He commented on obser-
the name mastzellen or mast cells (from the German verb vations by Gollasch that the sputum of asthma patients
mästen, meaning to fatten) because of his belief that mast contained CharcoteLeyden crystals and only eosinophils,9
cells occurred at sites with enhanced blood flow and and that a ‘material which attracts the eosinophils’ exists
nutrition. and on observations showing a ‘close connection’ between
When Ehrlich accepted the position at the Charité- the severity of asthma and eosinophilia and on similar
Universitätsmedizin in 1878, he turned his attention to observations in skin diseases. Ehrlich contended with
blood. He started with a modification of the simple theories that eosinophils are produced locally and argued
procedure that Koch used for the examination of bacteria in strenuously against this opinion while noting that mast cells
which blood or other fluid is spread as thinly as possible, are produced locally. To explain selective eosinophilia, he
rapidly dried at room temperature by exposure to air, and cited observations by Leichtenstern that blood eosinophilia
then stained. Hirsch and Hirsch comment that the use of diminished after a bacterial infection only to rise again
4 Eosinophils in Health and Disease

once the infection subsided.7 He stressed the importance of anaphylaxis.19 They examined the lungs of guinea pigs that
determining the absolute number of eosinophils as opposed survived anaphylaxis and described massive peribronchial
to the percentage and cited examples of how using just the eosinophilia. Because anaphylaxis in the guinea pig is often
percentages of eosinophils in the blood can lead the a lethal disease, it was reasonable to conjecture that the
observer astray. In a lecture given in Paris in 1900, Ehrlich presence of eosinophils is related to the prior anaphylactic
noted that ‘the leukocyte granulations are in fact secretory event. In 1931, Berger and Lang established that eosino-
products, which the cell dissolves and spreads to the phils also infiltrate the site of an immediate-type skin
environment as needed.’ In Histology of the Blood, Ehrlich reaction in passively sensitized humans.20 The concept that
and Lazarus state that ‘The final link of the chain of proof the eosinophil was associated with immediate-type,
of the secretory nature of the granules would be the direct anaphylactic sensitivity held sway for much of the 20th
observation of secretion by a granular cell.’7 Remarkably, century. This concept was tested by Archer and colleagues
this insight took almost a century to be realized and was who found that injection of an eosinophil extract and
shown by the release of eosinophil granule proteins with histamine diminished the intensity of the edema produced
a coating of tissues in various diseases, especially bronchial by intradermal histamine alone.21 In the absence of the
asthma13 and atopic dermatitis.14 eosinophil extract, a 10 mg histamine injection produced
However, functions of the eosinophilic leukocyte had to a wheal of 25 mm, whereas addition of eosinophils
wait characterization of the molecules comprising the cell, (between 0.8  106 and 25  106) reduced the wheal in
so that observations showing the release of granule proteins a dose-related manner to 10 mm. Observations such as this
in disease could be made, and the development of genetic seemed to point to the role of the eosinophil as a critical
molecular technology, so that animals with excessive cell for modulating inflammation, especially that mediated
numbers of eosinophils and animals devoid of eosinophils by histamine. However, in these experiments, controls
could be produced. testing the abilities of other cells to neutralize histamine
were not included. The concept of the eosinophil as an
anti-inflammatory cell was further investigated using bio-
EARLY DAYS: THE EOSINOPHIL AND chemical analyses and demonstrating that eosinophils
contain a histaminase and an arylsulfatase that might
ANAPHYLAXIS
function to degrade histamine and the slow-reacting
At the time of Ehrlich’s death in 1915, the procedures for substance of anaphylaxis [later characterized as leukotriene
determining the numbers of blood eosinophils, performed C4 (LTC4)].22 However, a direct test of the hypothesis that
much as he had described, and their associations with eosinophils function to modulate immediate hypersensi-
disease were established. Staining of tissues by hematox- tivity reactions conducted in guinea pigs, and abolishing
ylin and eosin became routine by the early 1900s,15 and the eosinophils with a specific antiserum failed to support this
associations between eosinophil tissue infiltration and hypothesis.23 The hypothesis that the eosinophil alters
diseases were further investigated, for example, the rela- tissues undergoing anaphylaxis, while not supported by
tionship between the occurrence of eosinophilia and asthma subsequent work on granule proteins, may yet be validated
in 1889 by Gollasch9 and between eosinophilia and trich- by current work on eosinophil cytokines, in that numerous
inosis in 1898 by Brown.16 However, the analyses of molecules produced by the eosinophil could affect local
complex mixtures of proteins using procedures that we tissue homeostasis.
today take as routine were primitive. This period of When I joined the Mayo Clinic staff in 1965, differen-
eosinophil research is well described by Samter.2 tial leukocyte counts were still being performed by staining
Among these advances, several stand out. The first was blood smears and counting 100e200 cells manually, and
the demonstration of marked eosinophil tissue infiltration Mayo employed about 30 workers for this purpose. In
in severe asthma.17,18 Huber and Koessler18 comment that essence, the technology was comparable to that introduced
the ‘coincidence of sputum and blood eosinophilia in the by Ehrlich 70 years earlier. In 1968, the seven-parameter
same individual seems to be a pathognomonic symptom of Coulter Counter Model S was introduced, and in the next
the asthmatic state’ and further state, ‘This tissue eosino- decade a series of innovations for automated blood count-
philia is a phenomenon of far-reaching bearing, which it ing proceeded. By the 1990s, automated counting was the
seems to us, if completely understood, would undoubtedly norm and the accuracy and precision of leukocyte counting
greatly elucidate the pathogenesis of asthma.’ The findings were markedly improved. Presently, counting of eosino-
of these authors17,18 corroborated the earlier work of phils is performed automatically and is ordinarily ordered
German workers showing that the presence of eosinophils by physicians as part of the complete blood count. Still,
and CharcoteLeyden crystals are characteristic of asthma. blood cell counts may be verified by visual inspection of
Another advance was the finding in 1912 by Schlecht and a blood smear. I am often struck by the failure of physicians
Schwenker of an association between eosinophilia and to order the enumeration of leukocytes in a differential cell
Chapter | 1 Historical Overview and Perspective on the Role of the Eosinophil in Health and Disease 5

count with the loss of information that might be helpful to themselves,36 basophils,37 neutrophils,38 and mast cells.39
their patients. They interact with other molecules such as the M2
muscarinic receptors,40 as well as clotting41 and comple-
ment42 components, altering their functions. They stimu-
EOSINOPHIL GRANULE PROTEINS AND late platelets with a potency comparable to thrombin,43
implicating the granule proteins as important molecules in
THEIR PROPERTIES clotting through platelet activation. This finding is likely
If the eosinophil does not have a dominant role as a cell relevant to the formation of thrombi and emboli in HES.
responsible for modulating hypersensitivity reactions, then The demonstration that the granule proteins possess RNase
what are its functions? During the late 1960s and extending activity, both RNase2 (EDN) and RNase3 (ECP),44 and that
for three decades, our laboratory at the Mayo Clinic they are able to neutralize viruses as a consequence of
investigated eosinophil granules and their proteins. These RNase activity45 suggest that eosinophils protect against
studies and others, particularly by Per Venge and virus infection. Further evidence for this capability came
colleagues,24 showed that the granule is composed of from studies of animal models.46
markedly cationic proteins, including eosinophil peroxi- Eosinophils and bronchial asthma have had an intimate
dase (EPO), eosinophil granule major basic protein 1 connection since their discovery. Once the granule
(MBP-1) and its homologue (MBP-2), and the eosinophil proteins were identified and became available, we used
ribonucleases, RNase2 [also referred to as eosinophil- them to probe their relationship with asthma. The sputum
derived neurotoxin (EDN)], and RNase 3 [the eosinophil from patients with asthma had elevated levels of MBP-1,47
cationic protein (ECP)].25 On a molar basis, MBP-1 is the and it was diffusely deposited on damaged bronchi in
predominant granule protein while EPO predominates on patients dying of asthma.13 A key experiment revealed that
a weight basis.26 Miller working with Palade found that the both EPO and MBP-1 provoked bronchospasm when
eosinophil granule has a distinctive morphology, which instilled into the lungs of monkeys, and MBP-1 induced
consists of a core (or crystalloid) with a regular two- bronchial hyperresponsiveness.48 Antibody to MBP-1 pre-
dimensional structure, as detected by electron microscopy, vented the development of bronchial hyperresponsiveness
and a matrix.27 MBP-1 is localized to the core of the in a guinea pig model of asthma.49,50 Finally, EPO and
granule28 and appeared to be its sole constituent,29 while MBP-1 stimulated the bronchial epithelium to synthesize
the other granule proteins localize to the matrix. The factors that alter structural cells and modify extracellular
cationic nature of the proteins at once explained the avidity matrix composition and turnover.51 Overall, these findings
of eosin, an acidic dye, for the eosinophil and also provided indicate that eosinophil granule proteins have the capacity
an explanation for certain eosinophil functions because the to produce many of the characteristic pathological changes
cationic granule proteins are toxins. For example, the seen in asthma.
granule proteins, well exemplified by MBP-1,30,31 are able While these discoveries related eosinophil granule
to kill helminths, such as the schistosomula of Schistosoma proteins to asthma, other work found that LTC4, which
mansoni,32 Trichinella larvae, and the microfilariae of powerfully contracts respiratory airways, is produced by
Brugia pahangi and Brugia malayi,33 and also damage eosinophils.52,53 These discoveries ultimately lead to the
tissues including the bronchial epithelium, keratinocytes, development of new therapies for asthma to prevent LTC4
pneumocytes, and the gut epithelium.34 The toxic granule formation or block its activity.54 Similarly, platelet-
proteins are deposited on tissues during the course of activating factor and prostaglandins are produced by
disease and coat the affected tissues.35 These observations eosinophils and likely are important in the pathophysiology
hark back to Ehrlich’s speculation that ‘the leukocyte of asthma.55
granulations are in fact secretory products, which the cell
dissolves and spreads to the environment as needed.’3
The links between diseases and eosinophil granule INTERLEUKIN-5, EOTAXIN, AND
protein deposition suggested a protective role in helminthic
diseases and an inimical role for the eosinophil in other
EOSINOPHIL-DERIVED CYTOKINES
diseases, such as asthma and hypereosinophilic syndrome With the development and application of recombinant
(HES). However, this evidence is only associative, and it DNA technology during the 1980s, it became possible to
was not possible to specifically enhance or ablate eosino- synthesize and purify proteins, such as cytokines, that are
phils in disease until more recently (see the sections on IL-5 present in such low concentrations that their isolation and
and transgenic and knockout mice later in this chapter). characterization are otherwise extremely difficult or virtu-
Continuing investigations of the granule proteins, again ally impossible. Moreover, cytokines have varied biological
using MBP-1 as an example, confirmed their toxicities, but activities, and it may be similarly difficult to ascribe a given
also showed that they activated cells including eosinophils activity to a particular purified molecule because it may
6 Eosinophils in Health and Disease

contain unrecognized impurities that mediate its biological IL-5 receptor showed that it shared molecular structures
activities. Therefore, the standard for the discovery and with the receptors for granulocyte-macrophage colony-
naming of cytokines, in general, is the discovery of the gene stimulating factor (GM-CSF) and IL-3.65,66 Presently, the
for the molecule and the expression of the protein free of clinical effects of monoclonal antibodies directed against
other cytokine contaminants. IL-5 was independently human IL-5 and against the alpha chain of the IL-5
discovered by three groups. The discoveries were based on receptor67 are under study and already have shown bene-
three different biological activities illustrating the pleio- ficial effects in the treatment of HES68 and bronchial
tropic activities of the molecule. asthma.69,70 However, regrettably, none of these drugs is
While each of these discoveries is of interest, the most presently registered for clinical use.
cogent instance is likely the identification of the factor Another key discovery during this time period was
responsible for eosinophilia following parasite infection, eotaxin.71 The infiltration of eosinophils into guinea pig
this association between eosinophilia and helminthic lung after anaphylaxis established in 1912 initially identi-
infection dating from Ehrlich’s time. Later, T lymphocytes fied the relationship between anaphylaxis and eosinophil
were implicated in the regulation of eosinophil produc- tissue infiltration,19 and this model was used to discover
tion,56 and soluble eosinophilopoietic factors were found to eotaxin as a chemokine that particularly attracts eosino-
be produced by murine lymphocytes.57 Sanderson and phils. Subsequently, three eotaxins have been described,
colleagues investigated a murine helminthic infection each derived from a different chromosomal gene but with
model58 and established a murine thymoma cell line that- sufficient three-dimensional similarity that they function
produced a factor causing eosinophil differentiation, through the same chemokine receptor, C-C chemokine
termed eosinophil differentiation factor (EDF).59 However, receptor type 3. For a time IL-5 was also thought to have
the factor possessing EDF activity also stimulated murine eosinophilotactic properties, but a direct test failed to
B lymphocytes,60 and, because of the difficulty obtaining demonstrate appreciable activity.72 IL-5 did function in
pure factors so that their biological activities could be concert with eotaxin to increase infiltrating eosinophils by
characterized, this finding raised the question whether the increasing the numbers of available circulating cells.
factor contained two proteins with two activities or whether Lastly, the eosinophil is able to produce numerous
one protein possessed both activities. Purification of the cytokines, including: C-C motif chemokine 5 (CCL5/
T-cell factor demonstrated that one factor had both activi- RANTES); eotaxin [C-C motif chemokine 11 (CCL11)],
ties, but the structure of the factor remained determined. eotaxin-2 [C-C motif chemokine 24 (CCL24)], and
Subsequently, Japanese investigators, led by Takatsu, eotaxin-3 [C-C motif chemokine 26 (CCL26)]; GM-CSF;
expressed the protein from cells possessing murine B-cell interferon gamma (IFN-g); IL-1 alpha, IL-2e6, IL-8e10,
stimulation activity.61 The gene sequence was unique and IL-12, and IL-16; macrophage inflammatory protein 1-alpha
was termed interleukin 5 (colony-stimulating factor, [MIP-1-alpha; C-C motif chemokine 3 (CCL3)]; nerve
eosinophil) IL5. A sequence from murine IL5 was then used growth factor (NGF); platelet-derived growth factor
to probe a human genomic library,62 resulting in the subunit B (PDGF subunit B); stem cell factor; transforming
isolation of the human counterpart of the murine B-cell growth factor alpha (TGF-a) and beta (TGF-b); and tumor
growth factor. Sequencing of the human IL5 gene showed necrosis factor (TNF-a) (reviewed in55). The capacity to
that it was homologous to the murine B-cell growth factor produce these factors opens new horizons for the eosinophil
and that the protein coded by the gene had exclusively in the instigation and control of physiological and patho-
eosinophilopoietic activity. Subsequently, IL5 was inde- logical processes, and these roles are yet to be thoroughly
pendently discovered by analyses of a murine T-helper cell defined.
clone that expressed several activities, including the abili-
ties to cause immunoglobulin A (IgA) enhancement63 and
eosinophil colony formation. A cDNA sequence was iso- TRANSGENIC AND KNOCKOUT MICE
lated from these cells by expression cloning, and a human FOR THE INVESTIGATION OF
clone was isolated by cross-hybridization. Sequencing of
the human and murine genes showed that they were iden-
EOSINOPHILS
tical to the previously isolated IL5 gene referred to earlier. Modification of the genetic makeup of experimental
Thus IL-5 was discovered by its biological activities as animals became a reality during the latter part of the 20th
a murine B-cell growth factor, as EDF, and as a human IgA- century with the production of transgenic and knockout
enhancing factor and eosinophil colony-stimulating factor. mice. These animals constitute an invaluable resource for
Subsequent studies showed that the B-cell growth factor the study of the eosinophil because of the unique capacities
activity is confined to mice64 and that human IL-5 to increase eosinophil production by the overexpression of
possesses predominately the ability to stimulate bone IL-573,74 and to abolish eosinophil production by either
marrow cells to produce eosinophils. Identification of the deleting a high-affinity transcription factor binding site,
Chapter | 1 Historical Overview and Perspective on the Role of the Eosinophil in Health and Disease 7

GATA,75 or by poisoning eosinophil production by EOSINOPHIL-ASSOCIATED DISEASES

coupling the promotor for EPO to the gene for diphtheria
toxin.76 The latter animal strain, termed PHIL, is essen- From the time of Ehrlich’s death until the 1970s, our
tially devoid of eosinophils. When sensitized with antigen, knowledge of eosinophil-associated diseases appeared to
PHIL fails to develop a characteristic feature of experi- change very little. The definition of HES and the subse-
mental asthma, namely bronchial hyperresponsiveness.76 quent investigations by Wolff and colleagues at the
PHIL also fails to produce the expected T-helper type 2 National Institutes of Health in the 1970s and 1980s
(Th2) cytokines, including IL-4 and IL-5, ordinarily seen in allowed the classification of patients with marked eosin-
this asthma model, and to accumulate T lymphocytes in the ophilia into a reasonably discrete group such that clinical
lung.77 Reconstitution of PHIL with eosinophils (and Th2- investigations could be done.80e82 A subset of HES
polarized ovalbumin-specific T cells) restores its ability to patients showed mucosal ulcers and appeared to have
accumulate pulmonary T cells and Th2 cytokines. There- a poor prognosis.83 During the 1990s, clonal lymphocytes
fore, the eosinophil appears able to control the accumula- producing IL-5 defined a HES subset.84,85 In the first
tion of T cells. This capability could be expressed in decade of the 21st century, treatment of HES patients with
numerous diseases and opens a new window on the possi- imatinib mesylate, introduced for the treatment of chronic
bilities for eosinophil participation in disease. myeloid leukemia, produced dramatic and durable
The studies on murine models of asthma, performed in responses,86 and a deletion on chromosome 4 defined
the laboratories of James J. Lee and Nancy Lee at the Mayo a kinase responsible for eosinophil proliferation.87,88
Clinic in Arizona, have also provided strong evidence that Elevated serum tryptase levels identified a subset of HES
the eosinophil has the capacity to inflict pulmonary damage patients with tissue fibrosis, poor prognosis, and imatinib
that closely mimicks that seen in severe clinical asthma.78 mesylate responsiveness.89
As noted above, PHIL failed to develop the characteristic During the 1980s and 1990s, Spanish toxic oil
airway hyperresponsiveness seen in wild-type animals. The syndrome90 and eosinophilia myalgia syndrome91 were
absence of the eosinophil in this experimental model discovered, the former as an epidemic linked to rapeseed
suggests that it is critical for the development of bronchial oil intended for industrial use and adulterated with aniline
hyperresponsiveness. James J. Lee and Nancy Lee devel- added to olive oil and sold in Madrid and environs, and the
oped another murine asthma model by creating a double latter to tainted tryptophan; diffuse fasciitis (Shulman
transgenic mouse expressing IL-5 systemically and syndrome), described in 1975, is often seen in these
eotaxin-2 (CCL24) in the respiratory epithelium.78 patients.92 Patients with episodic angioedema showed
Remarkably, these double transgenic mice developed striking eosinophilia and had cyclical elevations of IL-
pulmonary pathologies characteristic of severe asthma with 5.93,94 Patients with subcutaneous nodules, eosinophilia,
marked local eosinophil lung infiltration, epithelial rheumatism, dermatitis, and swelling (NERDS) appeared to
desquamation, and mucous hypersecretion, and structural constitute a novel syndrome.95 Eosinophilic esophagitis
remodeling with airway obstruction, subepithelial fibrosis, emerged as an entity related to food allergy and gastro-
airway smooth muscle hyperplasia, and striking meth- esophageal reflux disorder.96,97 Overall, these advances
acholine-induced airway responsiveness. Indeed, the mice broadened the scope of eosinophil-associated diseases to
showed such a dramatic sensitivity to methacholine that encompass patients with marked eosinophilia, and with
many died during challenge. These mice showed extensive novel cutaneous syndromes and gastrointestinal diseases.
eosinophil degranulation as judged by release of EPO and
MBP-1. A key experiment was then conducted mating the CONCLUSION AND FUTURE
double transgenic animals to PHIL; the resultant animals
PERSPECTIVES
showed neither eosinophil pulmonary infiltration nor
bronchial hyperresponsiveness or any of the other features The 1970s and 1980s were the decades of eosinophil
seen in the double transgenic. This powerful genetic granule proteins. They were isolated, characterized, and
experiment is difficult to refute and strongly implicates the used to investigate eosinophil-associated diseases. The
eosinophil as an inflammatory cell able to mediate most, if proteins are toxins, are deposited on damaged tissues in
not all, of the features of severe asthma. disease, and likely mediate damage. They activate
The role of the eosinophil in helminthic disease, numerous cells including other leukocytes, mast cells, and
namely a murine model of trichinosis, has suggested that lung epithelial cells, to secrete their mediators and to
the eosinophil, rather than being critical for parasite produce factors altering tissues. The 1990s was the decade
destruction, may actually be important for helminth of eosinophil cytokine production, and a remarkable
survival.79 This surprising result suggests that the helminth number of cytokines are attributable to the eosinophil.
is able to modify its behavior as a consequence of its These cytokines are able to mediate a vast number of
environment. changes in eosinophils themselves, in other leukocytes, and
8 Eosinophils in Health and Disease

in the cells of organs afflicted by eosinophil-associated participated in the critical studies of release of eosinophil granule
diseases. The past decade might be termed the era of proteins into tissues, Dan Lewis, Donald Wassom, Steven Ackerman,
mouse-manipulated models for eosinophil mechanisms in Evan Frigas, Larry Pease, David McKean, Frank Prendergast,
that mice essentially devoid of eosinophils showed the role Rosa Ten, Catherine Weiler, Joseph Butterfield, Kimm Hamann, Rob
Barker, Hirohito Kita and, my wife and colleague, Kristin Leiferman.
of the cell in disease models, especially asthma, and
revealed capabilities not heretofore suspected, especially
the ability to regulate the infiltration of T lymphocytes. It is
noteworthy that the ability of eosinophils to regulate T
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ACKNOWLEDGEMENTS 16. Brown TR. Studies on Trichinosis, with Especial Reference to the
Increase of the Eosinophilic Cells in the Blood and Muscle, the
My work was supported by the Mayo Foundation for 36 years and Origin of These Cells and Their Diagnostic Importance. The
allowed our group to commence and continue investigations of the Journal of Experimental Medicine 1898;3(3):315e47.
eosinophil and eosinophil-associated diseases. I was also supported by 17. Ellis A. The pathological anatomy of bronchial asthma. Am J Med
grants from the National Institute of Allergy and Infectious Diseases Sci 1908;136:407e29.
for over 35 years, and this support was critical for the expansion of 18. Huber H, Koessler KK. The pathology of bronchial asthma. Arch
granule protein studies, and the studies on eosinophilia myalgia Intern Med 1922;30:689e760.
syndrome and HES. Many colleagues contributed to the work from 19. Schlecht H, Schwenker G. Uber lokale Eosinophilie in der Bron-
our laboratory and merit mention, including David Loegering, who chien und in der Lunge anaphylaktischer Meershweinchen. Arch
was a substantial contributor to the asthma studies, Gail Kephart, who Exp Pathol Pharamacol 1912;68:163.
Chapter | 1 Historical Overview and Perspective on the Role of the Eosinophil in Health and Disease 9

20. Berger W, Lang FJ. Zur Histopathologie der idiosynkrasischen 39. Piliponsky AM, et al. Effects of eosinophils on mast cells: a new
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and eosinophil-derived neurotoxin by immunoelectron microscopy. mediate airway hyperresponsiveness, m(2) muscarinic receptor
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 Chapter 2

The Evolutionary Origins and Presence

of Eosinophils in Extant Species
Michael P. McGarry

INTRODUCTION INVERTEBRATE HEMOCYTES

Reason suggests that in metazoans of increasing The organ specialization evident in more complex metazoans
cellular/histological size and complexity, there need to is attended by a need for systemic mechanisms to integrate and
be some basic and common functions performed by coordinate intracorporeal structures and functions. This pre-
cellular elements that will be prerequisite to and sented opportunities for the selective benefit of motile cells
necessary for survival and reproduction. These include, that could adapt for these tasks. Some of these generic qualities
among others: were well defined by Brehelin and colleagues3 in their
description of blood cells of select species of insects. The
l systemic integration of function;
putative selective functions for their set of circulating cells
l nutrient availability;
included lineage elements for blood cells. Some had dense and
l respiratory gas exchange;
spherical cytoplasmic granules and other organelles, others
l separation from the environment;
were actively phagocytic, while yet others contributed to
l elimination of programmed cell death and metabolic
capsule formation. Although stains for light microscopy were
waste products; and
said not to be definitive, some of the cells had eosinophilic
l protection from injury and invasion.
granulation.3 These features reflect the qualities of blood cell
Evolutionary theories and the associated trees (i.e., clad- populations for the successful extant species of higher verte-
ograms) have been built, in part, based on the presumed brates. That is, there are specialized cells for coagulation
conservation of diverse features that confer competitive (hemostasis), protection from environmental irritants and
advantages within a population or environmental niche. pathogens (phagocytosis and elimination of foreign bodies),
More recently this has been complemented, and in most respiratory gas exchange, and for functions related to struc-
cases confirmed, by the more robust methods of gene tural development and maintenance (e.g., encapsulation).
sequencing and protein function similarities.1,2 One Complicating any efforts to categorize the various
feature that occurs in such seemingly unrelated species as lineages of circulating blood cells, or hemocytes, of inver-
insects and amphibians is the transformation of body form tebrates is the fact that differentiation often occurs in
known as metamorphosis. While generally identified with circulation. While some localization of hemocytopoiesis
morphological features, this hormone-driven phenomenon occurs, morphological variation and lineage plasticity4
(ecdysone and thyroxine for insects and amphibians, result in a variety of developmental morphologies for the
respectively) involves many more complex changes at the complement of cells that defies uniform definitions. In
biochemical, metabolic, physiological, cellular, and addition, many other metazoan invertebrate species also
histological levels. How these changes conferred selective contain cells in the hemolymph that, to a greater or lesser
advantages and contributed to the evolution of species is extent, contain granulation of varying dimensions. The
not defined, nor is it understood how vestiges of these majority of morphological descriptions is for cells exam-
processes may contribute to current issues of health and ined by electron microscopy and thus precludes description
disease. Furthermore, the function of eosinophil gran- of eosin affinity. However, the ultrastructural qualities and
ulocytes, or their precursors, in these transformational the variety of granule structures may be reflective of more
events may offer insights into both the evolution than one lineage of granulocyte, which is likely to include
of contemporary species as well as offer clues as to the an acidophilic class. Thus, descriptions exist in the litera-
role of these unique hemocytes in health and disease. ture of such cells in moths5 and a variety of other insects.3

Eosinophils in Health and Disease. http://dx.doi.org/10.1016/B978-0-12-394385-9.00002-X 13

Copyright Ó 2013 Elsevier Inc. All rights reserved.
14 Eosinophils in Health and Disease

They are also described in oysters6 and clams.7 Moreover, ascidians (subphylum: Urochordata), describe cells with
Stein and Cooper8 cite eosinophil granulocytes in annelid cytoplasmic electron-dense granules, some of which are
roundworms. Ravindranath reviews arthropod eosinophil acidophilic. The granulocytes participate in encapsulation
granulocytes9 and Andrew gives descriptive reference to of experimentally induced reactions to foreign bodies.
eosinophil granulocytes in a range of invertebrate species.10 Presumptive eosinophil granulocytes are described in the
The question of which invertebrate species is the best to Giemsa-stained peripheral blood of larval lampreys
exhibit wandering cells with acidophilic cytoplasmic (ammocetes) (superclass: Cyclostomata), presumed to be
granules in the hemolymph is not as important as the notion contemporary examples of early vertebrates.14 These
that they are, in fact, evolutionarily older than those authors conclude, however, that ‘. although lamprey and
representative vertebrate species that have definitive gran- mammalian eosinophil granulocytes share a limited
ulocytes of the distinct lineage which has come to be known number of morphological and functional features no
as the eosinophil granulocyte. It is difficult, if not impos- conclusive evidence exists in the findings to date for any
sible, then, to infer if any of these cell types represent evolutionary significance in their (sic) results.’ They also
primitive cells on the evolutionary path to the vertebrate quote Wittekind,15 who suggested the unreliability of
cell we collectively identify as the eosinophil (leukocyte). ‘Romanowsky-type dyes . to be a reliable indicator for
Biochemical and/or molecular confirmation of these any homology . simply reflect(ing) a basic chemical
eosinophilic granule-containing hemocytes as the evolu- similarity with which these stains react.’ However, as one
tionary precursors of vertebrate eosinophils are sketchy at progresses from cyclostomes to cartilaginous fish, studies
best. Eosinophil peroxidase is one of two myeloperoxidases in a representative species of cartilaginous fish, the dogfish
and is specific to eosinophils. Daiyasu and Toh11 con- shark, reveal definitive peripheral blood eosinophil gran-
structed a family association to explore the relationship ulocytesdwith an excentric, lobular nucleus and electron-
between these two peroxidases specifically associated with dense cytoplasmic granules.16
myeloid granulocytes. Based on protein sequence align- If some extant teleosts (bony fishes) may be considered
ment, an unrooted phylogenetic family tree was con- representative of those that may have been the initial
structed. Assessment of subfamily groups led them to candidates of this Class when they first appeared (circa
conclude that myeloperoxidase (neutrophil granulocytes) 450 million years ago), then the eosinophil granulocyte has
and eosinophil peroxidase diverged before the mammalian already achieved clear morphological lineage distinction.
divergence some 60e70 million years ago. However, these One might presume, therefore, that whatever selective
analyses were not robust enough to suggest where in earlier pressures may have contributed, or may have been exerted,
invertebrate/vertebrate evolution specialization of unique to result in its favorable retention/refinement in the
individual granulocyte lineages occurred. successful evolution of more complex and adapted species,
had already occurred. Light (Giemsa-stained) and electron
microscopy were used by Weinreb17 to describe eosinophil
VERTEBRATE EOSINOPHIL granulocytes which have an ‘ . eccentric nucleus, occu-
pying one-third to one-half of the cell ..’ The nucleus is
GRANULOCYTES
further described as perhaps being ‘ . indented, sausage-
Vertebrate eosinophil granulocytes have been recognized as shaped or bilobed ..’ The caveat is made that ‘ . unlike
a unique lineage for more than a century.12 They have been mammalian eosinophil granules . those in the teleost are
studied in representative species from all classes of verte- less distinct, smaller and less refractile.’ However, the
brates by light and electron microscopy. Much is known of granules ‘ . are relatively large . enclosing one or more
their development. All are part of populations with self- internal bodies . sharply delineated . are denser than the
renewal and are maintained within relatively stable ranges, matrix of the granule . large enough to distort the shape of
even after substantial blood loss, by a process of dynamic the granule.’ Similar descriptions have been made for the
compensatory proliferation. Nomenclature notwith- loach.18 The distinctions are not so clear in other teleosts.
standing, the eosinophil granulocyte of mammals is the cell Reite and Evensen19 describe the mast cell/eosinophil
defined as having a multilobed nucleus with cytoplasmic granule cell in an excellent review of inflammatory cells of
granulation that is readily stained by eosin, a normal teleost fish, proposing that the ‘rodlet cell [of salmonids
constituent of Romanowsky and Giemsa dye preparations. (sic)] may represent a type of eosinophil granulocyte,’
These granules frequently exhibit an internal crystalline resurfacing the question of lineage identity of the purported
structure by electron microscopy. Most, if not all, of these eosinophil granulocyte of these vertebrates. More recently,
features are evident in cells of the eosinophil lineage from with the zebrafish as the study subject, Lieschke and
all vertebrates. Some are even apparent in transitional colleagues20 have applied light and electron microscopy as
forms of other subphyla of Chordata. For example, Wright well as genomics and proteomics to an analysis of
and Cooper,13 in their studies of inflammatory reactions in granulocytes and macrophages in embryos and adults.
Chapter | 2 The Evolutionary Origins and Presence of Eosinophils in Extant Species 15

Eosinophil granulocytes were negative for myelo-

peroxidase, which typically is found in the primary gran- (A)
ules of neutrophils. Eosinophil granules were positive for
peroxidase. By catalytic domain analysis they determined
that:
‘zebrafish peroxidase lay basal to the 3 closely related
mammalian peroxidases (myeloperoxidase, eosinophil
peroxidase, and lactoperoxidase), thereby complicating the
naming of the zebrafish peroxidase . isolated. This
phylogeny suggests that the gene duplication and diversi-
fication that occurred in mammals to create these various
peroxidases occurred after the evolutionary divergence of
fish and tetrapods.20’
In perhaps a not unfamiliar commentary in a compre-
hensive review, Carradice and Lieschke21 summarize that
‘the function of this . [adult zebrafish-(sic)] . eosinophil
granulocyte has not been demonstrated, there are no
molecular markers for it, its ontogeny is not described, and
one must not infer function from nomenclature.’
Although debate may surround the exact character of
eosinophils from fish, no such concerns exist with the other (B)
classes of vertebrata. One of the most insightful descriptions
of amphibian blood and bone marrow, with specific refer-
ence to eosinophils, is given by Jordan.22 Some other
examples of light and electron microscopic descriptions of
eosinophil granulocytes of amphibians include work by
Turpen and colleagues23 and Frank24,25 for frogs, as well as
Tooze and Davies26 and Hightower and Haar27 for newts.
Granulocytes with eosin-positive cytoplasmic granules were
typically identified; eosinophils were identified by electron
microscopy, but crystalloid bodies within the granules were
not consistent findings. However, there seems to be no
ambiguity of their lineage identity in both premetamorphic
and postmetamorphic frogs. This is also true for the
description of eosinophil granulocytes in the Class: Aves.
That is, both light and electron microscopic morphology of FIGURE 2.1 (A) Light photomicrograph of Romanowski-type stain of
eosinophil granulocytes of many avian species have been femoral marrow from a mature C57BL/6 mouse. Acidophilic granules,
classically defined by Maxwell28,29,30 and display an unde- typical of mammalian eosinophils are evident in two cells; cells of other
niable similarity with mammalian eosinophils. hemocytopoietic lineages are apparent. Morphologies and staining quali-
Descriptions of mammalian eosinophils are readily ties are typical of most mammalian species, variations owing predomi-
nantly to granule size. (B) Electron micrograph of an eosinophil myelocyte
available in any number of classical texts (see, for example, from femoral marrow of a C57BL/6 mouse that exhibits the characteristic
31). In addition, the light microscopic similarities of internal crystal structure of the specific cytoplasmic eosinophil granule. Its
eosinophils in Giemsa, Wright, MayeGrünwaldeGiemsa presence, considered definitive of the eosinophil lineage, is evident in
and Domenici-stained peripheral blood and marrow prep- mammals and several other non-mammalian species.
arations of many mammalian species are striking (M. P. M.;
unpublished observations) This has included the yellow
and olive baboon, bat, cat, cow, deer, dog, goat, guinea
COMPARATIVE EOSINOPHILOPOIESIS
pig, hamster, horse, monkeysdrhesus and stump-tailed There are many attractive comparisons of hemocytopoiesis
macaquesdpig, rabbit, and rat, as well as several inbred, across the metazoan continuum. Even primitive inverte-
hybrid, random-bred, wild, mutant, and genetically altered brates have hemocytopoiesis restricted to limited tissue
strains of the laboratory mouse (see Fig. 2.1). It continues sites.32 Ghiretti-Magaldi and colleagues33 concluded from
to be an enigmatic cell as to its place both functionally their observations in the common littoral crab Carcinus
and as an embedded clue to the evolution of higher maenas that three granulated and one agranular circulating
vertebrates. hemocytes derive from a single, common stem cell. In
16 Eosinophils in Health and Disease

vertebrates, the sites of hemocytopoiesis in general and exchange occurs in newly developed lungs as the
eosinophilopoiesis in particular are especially conserved amphibian adapts from an aquatic to a terrestrial environ-
and are generally restricted to specialized organs. Even in ment. These changes are dramatic and require metabolic,
the most primitive protochordates in which it has been physiological, and anatomical alterations (i.e., remodel-
studied, hemocytopoiesis is organ-specific. ing). The role of eosinophils in these processes is not well
As one ascends the vertebrate evolutionary tree, eosi- defined, but warrants study. The evolutionary importance of
nophilopoiesis is found progressively in the liver, intestine, the eosinophil may well be rooted in the selective advan-
kidney, adrenal gland, thymus, spleen, and bone marrow tages conferred on evolving species through its beneficial
(see 10). In mammals, this progression is followed devel- role in metamorphosis. Interestingly, Rheuben5 observed
opmentally, beginning in fetal liver, moving to the spleen, large numbers of phagocytic granulocytic hemocytes
and eventually becoming established in the marrow of most accumulated on the surface of degenerating muscle fibers
of the bones of the mature body. Extramedullary hemocy- during the prepupal-to-first-day postecdysis in the moth,
topoiesis in mammals under stress (e.g., of excessive blood Manduca sexta. That is, granule-containing hemocytes are
loss or other conditions related to overproduction or associated with tissue remodeling at metamorphosis in
pathology) usually occurs in these developmental/more invertebrates!
primitive hemocytopoietic sites. Analysis of the eukaryotic orthologous groups (KOGs)
of proteins has been applied to the dilemma of whether the
foundational roots for the evolution of metazoans is based
SO, WHY EOSINOPHIL on Coelomata (with a true body cavity, e.g., arthropods and
chordates) or Ecdysozoa (molting animals).38 Although
GRANULOCYTES? considerable controversy still prevails in the interpretation
The role and/or function of the eosinophil granulocyte in of data, suffice it to say that a significant base in the
the vertebrate body have resisted definition. The arguments successful evolution of more complex metazoans occurred
for a reconsideration of the classical understanding of as earlier ancestors of contemporary vertebrates encoun-
this were recently put forward by Lee and colleagues.34 It tered selective pressures for survival. Either successive
was proposed that ‘. accumulating tissue eosinophils are instars, which yielded intermediate periods for increased
actually regulators of Local Immunity And/or Remodeling/ body growth among other refinements (Ecdysozoa), or
Repair in both health and diseasedthe LIAR hypothesis.’ possession of a body cavity, perhaps conferring advantages
In it, the case is made that the role of eosinophils is not in organogenesis and anatomical composition (Coelomata),
primarily in host defense. While there is ample evidence would be the determining structural advantage for the
that these cells are well equipped for contributing to the continued progressive evolution to early vertebrates. From
elimination or containment of metazoan parasites and other that period through the emergence of reptiles, a midlife
select pathogens, consideration of other responses and the metamorphosis characterizes successfully evolved, more
timing of their appearance in the evolutionary continuum complex species. This process coincidently also reflects
would make it unreasonable to associate the eosinophil a substantial refinement of blood cell differentiation,
exclusively with conferring a selective advantage in the especially as regards granulocytes and specifically the
defense against parasitic infestation. Even the process of eosinophil. Primitive chordates also undergo a meta-
disgorging granule contents (i.e., degranulation) is not morphosis.39 It seems reasonable to propose that evolution
a consistently conserved function of all eosinophils in host of higher vertebrates resulted from the successful adapta-
defense mechanisms.35 It is thus doubtful that this feature tion of early vertebrate species that included a considerable
was a dominant determinant of a selectively advantageous body makeover, requiring remodeling events. Thus, could it
function of evolving eosinophil granulocytes. In the LIAR be that residual functions of eosinophils have their roots in
hypothesis, eosinophil effector functions are proposed to be the breakdown and reconstruction of histological struc-
more closely related to remodeling events. The authors tures, especially those related to respiration and digestion?
reference the observations of Jordan and Speidel36 that the It is enigmatic that the eosinophil granulocyte, so
dramatic shortening of the frog tadpole gut at meta- ubiquitous in vertebrate organisms, is still without a firm,
morphosis [M. P. M.; unpublished observation (see also37)] exclusive function. Moreover, sophisticated genetic tech-
is accompanied by a substantial infiltration of eosinophils. nologies have made it possible to extinguish the eosinophil
The metamorphosis of anuran amphibians is an elaborate lineage from at least one mammalian species, the mouse.40
process involving substantial histological and physiological One might have anticipated that the absence of the lineage
changes: absorption of the extensive tadpole tail, develop- would result in some developmental or functional defi-
ment of complex multicellular skin glands, the aforemen- ciency so as to make survival impossible. However, at least
tioned gut alterations as the animal changes from under the protective custody of contemporary husbandry
a herbivore to a carnivore, and gill resorption as gas for laboratory animals, no life-threatening abnormalities