Eosinophils: Methods and Protocols

1. Eosinophil Overview - Structure, Biological Properties And Key Functions Paige Lacy, Helene F. Rosenberg, Garry M. Walsh 2. Eosinophil Purification From Peripheral Blood Praveen Akuthota, Kelsey Capron And Peter F. Weller 3. Eosinophil Purification From Human Bone Marrow Tina W. Wong And Diane F. Jelinek 4. CD34(+) Eosinophil-Lineage-Committed Cells In The Mouse Lung Apostolos Bossios And Madeleine Radinger 5. Eosinophil Cell Lines Kenji Ishihara 6. Cell Signalling In Eosinophil During Human Eosinophil Differentiation Miranda Buitenhuis 7. Eosinophil Intracellular Signalling - Apoptosis Pinja Ilmarinen, Eeva Moilanen And Hannu Kankaanranta 8. Identification Of Human Eosinophils In Whole Blood By Flow Cytometry Caroline Ethier, Paige Lacy And Francis Davoine 9. Isolation And Functional Assessment Of Eosinophil Crystalloid Granules Renata Baptista-Dos-Reis, Valdirene S. Muniz And Josiane S. Neves 10. Eosinophil Chemotaxis Gordon Dent 11. Human Eosinophil Shape Change And Secretion Lian Willetts And Paige Lacy 12. Human Eosinophil Adhesion And Receptor Expression Colleen S. Curran 13. Adhesion Of Eosinophils To Endothelial Cells Or Substrates Under Flow Conditions Viktoria Konya, Miriam Peinhaupt And Akos Heinemann 14. Human Eosinophil Transmigration Stanislawa Bazan-Socha, Joanna Zuk, Bogdan Jakiela And Jacek Musial 15. Measurement Of Eosinophil Kinetics In Healthy Volunteers Neda Farahi, Chrystalla Loutsios, Rosalind P. Simmonds, Linsey Porter, Daniel Gillett, Sarah Heard, A. Michael Peters, Alison M. Condliffe And Edwin R. Chilvers 16. Assays Of Eosinophil Apoptosis And Phagocytic Uptake David A. Dorward, Sidharth Sharma, Ana L. Alessandri, Adriano G. Rossi, Christopher D. Lucas 17. Clinical Measurement Of Eosinophil Numbers In Eosinophilic Conjunctivitis Osmo Kari And K. Matti Saari 18. Qualitative And Quantitative Studies Of Eosinophils In Parasitic Infections Masataka Korenaga And Fabrizio Bruschi 19. Interactions Of Eosinophils With Nerves Quinn R. Roth-Carter, David B. Jacoby And Zhenying Nie 20. Eosinophils Interaction With Mast Cells: The Allergic Effector Unit Roopesh Singh Gangwar And Francesca Levi-Schaffer 21. Eosinophil Interactions: Antigen Presentation Praveen Akuthota 22. Eosinophils And Respiratory Virus Infection: A Dual-Standard Curve Qrt-PCR-Based Method For Determining Virus Recovery From Mouse Lung Tissue Caroline M. Percopo, Kimberly D. Dyer, Kendal A. Karpe, Joseph B. Domachowske And Helene F. Rosenberg 23. Antimicrobial Activity Of Human Eosinophil Granule Proteins Anu Chopra, And Janendra K. Batra 24. Eosinophils And The Ovalbumin Mouse Model Of Asthma F. Daubeuf And N. Frossard 25. Mutant Mice And Animal Models Of Allergic Disease Marie-Renee Blanchet, Matthew Gold And Kelly M Mcnagny 26. Murine Models Of Eosinophilic Leukemia - A Model Of FIP1L1-PDGFRa Initiated Chronic Eosinophilic Leukemia/Systemic Mastocytosis Yoshiyuki Yamada, Jose A. Cancelas And Marc E. Rothenberg

The peripheral blood eosinophilic ' sukocyte was first described by Whar-' on-Joncs in 1846 as a coarse granular ^"dl in contradistinction to the less uni-' orm and more finely granular poly-"lorphonuclear neutrophil. With the advent of more advanced histological techniques. Paul Ehriich in 1879 noted remarkable affinity of the granules °f this cell for the acid dye eosin and ^feby provided a name for this dis-•^'vision of Allergy and Clinical Im-" 'unology. tinctive cell. As viewed in the peripheral blood, the eosinophil has a diameter of 15-20 microns. Its nucleus is most frequently bilobed although multilobed forms may be seen. The most distinguishing feature, however, is the numerous 0.3-0.7 micron uniform spherical granules which fill almost Ihe entire cytoplasm of the cell.
The bone marrow appears to be Ihe only site of eosinophil formation. Development from myeloblast to mature cell occurs within 24-72 hours. After reaching maturity the cell remains with-209 in the bone marrow for three to four days before prtKceding to various tissue sites. Although the peripheral blood IS frequently used as a sampling site for evaluation of eosinophil levels, this cell is predominantly a tissue cell and appears to utilize the blood stream primarily as a means of transport. Onehalf of the eosinophils leave the blood stream on their first passage through the circulatory system; most have entered the tissues within one hour of their release from the marrow. The bone marrow represents the primary tissue storehouse for eosinophils; other tissues with significant numbers include the lung, intestinal tract and skin. The total life span of this cell in the marrow, bloodstream and tissues has been calculated to be between 8 and 12 days.
While the peripheral blood may not always acuratcly reflect total body levels of eosinophils, differential counts of peripheral blood leukocytes or the more accurate total peripheral blood eosinophil count remain the most frequently used means of detecting disturbances in eosinophil numbers. Normal levels of eosinophils range between SI) and 250 cells per cubic millimeter. Normal counts show diurnal variation with the highest levels being reached during the evening hours. Since steroid htirmones are known to produce cosinopenia (this eflect was the basis for the original Thorn test of adrenal function), the normal circadian rhythm of steroid hormones has been utilized as an explanation for this normal variation in eosinophil levels. Disease states asscKiated with disturbance in eosinophil numbers are usually characterized by markedly increased numbers of these cells within the peripheral circula-tion. These disorders represent a diverse array of morbid conditions which thus far have appeared to lack a common denominator which would explain their association with eosinophilia. Such a circumstance is not too surprising, however, when one considers that despite 100 years of study, the precise function of this distinctive cell within the normal body economy is completely unknown. A brief review of some of the conditions frequently associated with eosinophilia and of some of the more recent experimental work related to eosinophil function may serve to highlight our present state of knowledge about this cell.

Conditions Frequently Associated
Willi Eosiiuiphilia: Remarkable degrees of eosinophilia (up to 85% of circulating WBC's) have long been known to occur in a variety of parasitic infestations includ ing infections with protozoa, roundworms and flatworms. The most in tense eosinophilia occurs at the time of direct tissue invasion by the parasite While an "allergic" mechanism of some sort is often invoked as an explanation for the presence of eosinophils, the precise means by which these cells arc attracted in such great numbers is n<^ at ;ill clear.
A relatively distinct clinical entii} found in warm countries bears the not unexpected apellation of tropin' eosinophUia. This disorder consists marily of pulmonary infiltrates anJ striking blood eosinophdia. Because oi its response to Hetrazan, the disei^ has long been suspected to be an un usual response to filaria. The parasite however, has not yet been defflc"' strated and the etiology remains unknown. Even if a causative organism could be found, the mechanism of the eosinophilia would still require explanation.
A variety of clinical disorders resembling tropical eosinophilia have been described in temperate climates and are usually lumped together under the descriptive term, P.I.E. syndrome (Pulmonary Infiltrates and Eosinophilia). Some of these are acute and transient like the fleeting pulmonary shadows initially described by Loeffler and occurring predominantly in patients with bronchial asthma, while others tend to have a more granulomatous character. The variation in severity and association with a variety of accompanying conditions (asthma, infection, exposure to drugs, etc.) suggest that a common etiologic agent is not operative and that the P.I.E. syndrome as a whole represents a heterogeneous group of conditions. Again the mechani >m of the eosinophilia is unknown.
The collagen diseases represent another group of clinical disorders which may be associated with eosinophilia. TTi i s is especially exemplified by poly-Weritis nodosa in which increased i' vels of circulating eosinophils are 'en in about 30% of cases. These disorders share a suspected autoim-•' lune etiology which some feel could ^r\e as well to explain the presence of •osinophils. The precise mechanism the eosinophilia. however, seems no I i^re clear than the relatively unprecise autoimmune" nature of the basic dis-Tder.
Eosinophilia has been described in a *^'y of neoplastic diseases. In cer-Malignancies such as Hodgkins and eosinophilic leukemia. eosinophils appear to be an integral part of the cellular infiltrates. Like various allergic reactions such as urticaria, the eosinophilia in solid tumors has been felt to be related to necrosis within the tumor itself-which could result in the release of tumor antigens and a subsequent antibody respon.se on the part of the patient.
In some patients eosinophilia may exist as an apparently benign finding without evident cau.se. The occurrence of this finding within several members of the same family has led to the description of so-called familial eosinophilia. While some feel that certain examples of this disorder are related to occult parasites, such as visceral larva migrans, extensive studies have failed to reveal a specific underlying cause in other instances.
For many years pathologists have emphasized that the eosinophil can be commonly seen in a variety of cellular infiltrates, particularly chronic ones. Without special staining procedures, eosinophils may be difficult to distinguish in tissue sections. This may explain the failure lo appreciate their presence even when in considerable numbers. In effect this suggests that the eosinophil, like the plasma cell, may function in certain instances as a chronic inflammatory cell.
Eosinophils have long been of special interest to physicians dealing with allergic disorders. Witness the copious numbers of these cells in Ihe nasal secretions of patients with hay fever, the bronchial secretions of those with asthma and in the blood in both of these conditions as well as in atopic dermatitis, drug reactions and urticaria. Indeed, the consistent finding of both tissue and blood eosinophilia accompany-ing allergic responses has led many to equate eosinophilia with the presence of an underlying allergic reaction. Eosinophils certainly arc present in allergic reactions. However, they are frequently present in as great or even greater numbers in clinical circumstances in which even careful study fails to demonstrate any evidence of allergy. In many patients with nasal polyps and by definition in patients with intrinsic bronchial asthma, no evidence of underlying hypersensitivity can be found, yet the blood and secretions of these individuals regularly reveal striking numbers of eosinophils. This suggests that in such instances eosinophilia is related to a more basic defect in the diseased individual rather than being a simple accompaniment to an allergic reaction.
Even this brief perusal of clinical conditions associated with eosinophilia emphasizes the diverse disorders which may underlie the presence of increased numbers of this distinctive leukocyte. A fundamental question in each instance must be; "What has stimulated the increased production of eosinophils by the bone marrow and Ihe attraction of these cells to specific tissue sites-i.e. what is the mechanism of cosinophdotaxis?" An even more basic question to be considered is: "Once out of the txmc marrow, what is the precise function or functions of this cell?" I osin()|»liil(il.i\is I he demimstration of large numbers of eosinophils, in biopsies of positive skin tests performed on allergic subjects, suggests that allergens may function as eosinophilotactic substances in atopic individuals. This phenomenon has been investigated further by mei-ns of the Rebuck skin window tc inique.' ^ The application of pollen antigen to skin windows results in the release of numerous eosinophils into the infiammatory exudate. The exact nature of the eosinophilotactic substances is uncertain. Most agree that histamine, released during the course of allergenreagin combination, does not attract eosinophils.-"'" In analogy to the work of Litt, described later, the allcr;enreagin complex itself has been felt to be the prime eosinophilotactic substance. However, the possibility exists that other materials generated during Uie allergic reaction might be important chemotactic substances. For example. Riddle' has demonstrated that the dog eosinophil granules contain profibnnolysin and that fibrin in this species is eosinophilotactic. This has led one group of workers to emphasize tbe presence of fibrin in various allergic reactions (allergen challenged skin windows and nasal polyps) and lo sue gest that fibrin may play a chemotactic role for eosinophils in allergic reactions.^ Note also that this thesis suggests a specific role for the eosinophil in infiammatory states-that of proM^! ing fibrinolysin.
In a series of elegant experiment" Litt '' has demonstrated the eosinophiK^ tactic ability of IgG antigcn-antibod} complexes in the intact guinea pig-T' ' ' " lowing the intraperitoneal injection ^ antigen-antibody complexes prepare^m vitro, eosinophils not only migrate^ into the peritoneal cavity but al^ i phagocytized the complexes. Furthfmore, there was demonstrable degra"-ofit< lation of eosinophils in the area cytoplasm occupied by the comple^^ These experiments likewise suggest a specific role for the eosinophil -that of phagocytizing antigen-antibody complexes.
Ward,-' however, has recently emph;.->ized that the eosinophil responds to substances (soluble bacterial factors in culture filtrates and activated components of complement) known to be chemotactic to neutrophils. It would appear therefore that all eosinophilotactic stimuli need not be specific; it is possible that IgG antigen-antibody complexes might attract eosinophils by virtue of their ability to activate the complement system. This possible mechanism would not apply to the allergcn-reagin complex or to IgA antigen-antibody complexes since IgE and IgA antibodies do not fix complement. Also. Zolov and Levine'" have noted that eosinophilia developing in patieuis rcceiMug penicillin correlates best with the presence of skin sensitizing antibody (IgE) rather than with IgG or IgM antibodies directed against antigenic derivatives of penicillin.
Liu" has also observed the apparent eosinophilotactic action of antigen. \Vithin five minutes of injection of antigen into the foot pad of guinea pigs, eosinophils were observed to diapedese -hrough the blood vessels of draining ' ymph nodes and within a few hours numerous eosinophils were present -round the medullary cords of the "•ode. Liu interpreted this as indicating ' apid synthesis of antibody by the ^"iph node cells and the formation antigen-antibody complexes which l^' as the actual chemotactic substance.
°berts'-has suggested that antigen * may be responsible for the ata of I** I-Taction of eosinophils. She demon-nple^^ 11 '^^'tl the presence of radioactively labelled antigen within eosinophils of draining lymph nodes four hours after antigen injection. However, her studies did not rule out the possibility that the antigen was complexed with specific antibody. Cohen" on the other hand has felt that in many instances eosinophils may be attracted by non-inmiunologic stimuli. He demonstrated that numerous substances, including polystyrene particles, can be eosinophilotactic.
The question of whether there exists a specific eosinophilotactic stimulus still remains unanswered. Some work strongly suggests that eosinophils may be attracted by non-immunologic stimuli and that their presence docs not necessarily indicate a respi)nsc to a stimulus specific for that particular leukocyte.
Another possible explanation for the presence of eosinophilia in certain clinical situations comes from a recent theory concerning the etiology of bronchial asthma." This theory proposes that the basic underlying defect in asthma is a partial blockade of the betaadrenergic receptor sites in the bronchial tree. Since stimulation of the beta-adrenergic portion of the sympathetic nervous system is known to result in eosinopenia, a blockade of these sites would be expected to result in eosinophilia. Such a mechanism could serve to explain the puzzling finding of significant eosinophilia in clinical conditions usually of allergic origin but in which no evidence of underlying allergy can be found. In such instances the number of eosinophils is controlled primarily by the sympathetic nervous system without demanding the presence of allergen-reagin or other eosinophilotactic stimuli. 213 Sweet Eosinophil Function Experimental data have suggested two possible functions for the eosinophil-the phagocytosis of antigen and antigen-antibody complexes and the transport of fibrinolysin to areas of infiammation. .Additional studies concerning the phagocytic ability of the eosinophil have suggested that they may ingest a variety of particulate substances (bacteria, fungi, polystyrene particles. RBC's complexed with antibody, mast cell granules) but that in general they are less efficient in this function than the neutrophil. It would therefore seem unlikely that phagocytosis is a major raison d'etre for the eosinophil.
The association of eosinophils with allergic reactions raises the possibility that this cell might be responsible for the transporlatitm of histamine (a major mediator of immediate hypersensitivity reactions) or that it might function to bring antihistaminic substances to the site of allergic reactions. Analysis of the cytoplasmic contents of eosinophils has revealed small amounts of both histamine and a substance with slight antihistaminic activity. However, the amounts present seem so small that they are unlikely to contribute significantly to the role of the cell." l-nzyme analysis of eosinophilic granules has revealed the presence of a number of potent enzymes including cathepsin, ribonuclease, aryKulphatase, Ix-la-glucuronidase. acid phosphatase, alkaline phosphatase and peroxidase." I xcepi fi^r the relatively high content of peroxidase, the enzyme composition is quite similar to that found in the granules of the neutrophil. The eosinophilic granules can therefore be considered lysosomes. The necessity for such potent enzymes is not entirely clear although they could act upon ingested particulate (bacteria, fun-ji) or soluble (antigen, antigen-antibody complexes) material.
The eosinophil along with the basophil recently has been shown to be the source of an unusual structure known for years as the Charcot-Leyden crystal."* First described by Charcot and Robin in 1843 in the spleen of a patient with chronic leukemia, these crystals have the appearance of two hexagonal pyramids joined at the base Twenty-nine years later Leyden noted similar structures in sputum from ,i patient with bronchial asthma. The spontaneous formation of these crystals within the cytoplasm of human eosinophils kept in saline for 24 hours was finally observed by Archer and Blackwood in 1965. The crystals appear to be protein in nature and probably result from degenerative changes within the cytoplasm.
Thus, though our knowledge concerning the eosinophil has increased re markably since 1846, many of its tunc tions require future explanation. Further study of eosinophdotactic stimuli and eosinophil function should ser\e to characterize the precise roles of the eosinophil in the body economy. Inter est in this distinctive leukocyte exisi> in a variety of medical disciplines an<: greater understanding may be anti»-ipated before another 123 years ha^e passed.