Histol Histopath (1994) 9: 339-374 Histology and Histopathology

Invited Re vie W

Human in vitro. An ultrastructural morphology primer

A.M. ~vorakland T. lshizaka2 'Departments of Pathology, Beth lsrael Hospital and Harvard Medical School and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Masachusetts, and *Division of Allergy, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

Summary. An ultrastructural morphological primer of functioning rnenibers of other lineages, also present in c- human eosinophils is presented. Mature and immature kit ligand-supplemented cultures. These lineages include eosinophils. obtained from peripheral blood and bone mast cells, , . monocytes, macro- marrow, as well as activated tissue eosinophils are all phages. , and endothelial cells. used to illustrate the various morphologies assumed by eosinophils in vivo. The various ultrastructural changes Key words: Human, Eosinophils, In vitro, Ultrastri~cture, expressed by this cell lineage in vivo reflect the impact of Growth factors differentiation, maturation, activation, secretion, and cell injury on morphology. Nearly all of the changes described in vivo are also evident in eosinophils arising 1. Introduction in in vitro systems. We review published studies of these culture systems, which have been supplemented with The rapid identification of hematopoietic growth various conditioned media containing naturally factors and culture systems, which specifically enable occurring growth factor(s) that are permissive (or not the development of the lineage, has pro\4ded permissive) for eosinophils or with the recombinant an opportunity to learn more about the morphologic growth factors, IL-5 or IL-3. These sti~dieswere helpful expressions of these during their in the recognition of eosinophil-promoting, -sustaining maturation and function in vitro (Dvorak et al.. 1993a). and -activating properties of human IL-3 and IL-5. The alterations in the standard, well-known ultra- Moreover, mature and immature eosinophils were shown structural morphology of normal human peripheral blood to release a matrix protein - eosinophil eosinophils (Dvorak et al., 1991a) that are observed peroxidase (EPO) - by its transport in small cytoplasmic in these culture-derived cells are considerable. vesicles, a process termed piecemeal degranulation Additionally, depending on the growth factor(s) present, (PMD). accounting for the gradual emptying of granule eosinophils are found among representatives of other contents in the absence of granule fusions to the plasma cell lineages. These cells are also undergoing membrane. Also presented are eosinophil morphologies morphological changes secondary to their developmental that occur in vitro in suspension cultures of human cord and functional programs. Therefore, the ultrastructural blood supplemented with the c-kit ligand from various identification of members of the eosinophil lineage is sources. The wide variety of eosinophil subcellular difficult. For this purpose, a primer of ultrastructural changes in the c-kit ligand-supplemented cultures, like eosinophil morphologies in vitro may be useful to the the changes of which eosinophils are capable in vivo, research community. reflects the processes of differentiation, maturation, We review here, briefly, the ultrastructural activation, secretion and cell injury. Presentation of this morphology of normal eosinophils in peripheral blood. ultrastructural morphological primer of human of immature eosinophils in , and of eosinophils in vitro should enable investigators to functioning tissue eosinophils in vivo. More complete recognize eosinophils in all of their diverse morphologic ultrastructural reviews of these in vivo morphologies forms in cultures that contain differentiating and have recently been published (Dvorak, 1988, 199 l b. 1993; Dvorak et al., 1991a, 1993a; Weller and Dvorak, Offprint requests to: Ann M. Dvorak. M.D.. Department of Pathology. 1994). We also review recent reports of in vitro systems Beth lsrael Hospital. 330 Brookline Avenue. Boston. MA 02215, USA that reflect some ~~ltrastructuralaspects of mature and Morphology of human eosinophils in vitro

immature eosinophils and their functional rnorphologies cytoplasmic processes. The cytoplasmic (Fig. (Dvorak et al., 1985, 1989, 1991b. 1992. 1993f; Jabara 2) include glycogen particles, secretory granules. lipid et al., 1988; Saito et al., 1988). Newer culture systems, bodies and tubulovesicular structures. Golgi structures supplemented with fibroblast products, also contain and membrane-bound ribosomes are diminished. The eosinophils, generally as a minor population in early large, round lipid bodies are osrniophilic and are not cultures (Dvorak et al., 1993b,c,e. 1994a.b; Mitsui et al., bound by a trilaminar unit membrane. They may be 1993). These suspension cultures provide a challenge to completely or focally encircled by a single. membrane- the ultrastructural morphologist that shoi~ldbe facilitated like dense shell. Focal, internal electron-lucent areas and by this documentation of the various morphologies dense particles can be observed within lipid bodies. and expressed by eosinophils in vitro. Thus, identification individual lipid bodies may fuse. Lipid bodies are criteria are presented for mature eosinophils that are arachidonic acid-rich organelles (Weller and Dvorak. granule-poor or granule-free. that contain lipid bodies 1985: Weller et al., 1991a) that contain the enzyme only, that have increases in small granules and primary prostaglandin endoperoxide synthase. as demonstrated granules, and that have a variety of alterations in their by ultrastructural autoradiography and immunogold major bicompartmental granule population. These stains, respectively (Weller and Dvorak, 1985; Weller et ultrastructural images should make it possible to al., 1991a,b; Dvorak et al.. 1994~).The majority of recognize members of the eosinophil lineage and granules in mature eosinophils are large, membrane- to distinguish them from other cellular lineages cound. oval-shaped structures within which a central, simultaneously present. These include mast cells. dense, crystalline core compartriient and an outer. less basophils, neutrophils, monocytes. macrophages, dense matrix compartment are seen (Fig. 2). These megakaryocytes and endothelial cells and their immature are secondary or specific granules. In their core andlor functional forms (Dvorak et al., 1993b,c,e, compartment is found (MBP): 1994a.b; Mitsui et al., 1993). (EPO), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), 2. Mature human eosinophils and tumor necrosis factor-alpha (TNF-a) are found in their matrix compartment by i~ltrastructuralimrn~~nogold Mature eosinophils (Fig. I) are granulocytes that and cytochemical methods (Egesten et al., 1986; Peters have multiple lobes to their nuclei. The nuclear et al.. 1986; Dvorak et al., 1991a, 1993a; Beil et al., chromatin is condensed, and nucleoli are absent. The cell 1993). Approximately 5% of the granules lack central surface contours are comprised of irregular broad cores; these are residual primary granules (Fig. 2), which

Fig. 1. A mature eosinophil (small intestinal biopsy) shows two nuclear lobes with condensed peripheral chromatin, irregular, broad surface processes and secretory granules. Bicompartmental secondary granules (open arrowhead), a single prlmary granule (closed arrowhead), an osmio- philic lipid body (arrow), cytoplasmic vesicles and mitochondr~aare present. X 16,500 Morphology of human eosinophils in vitro

have been determined as the granule storage condensetl chromatin along nuclear membranes. for the Chnrcot-Leyden crystal protein by ultrastructural Cytoplasmic glycogen and lipid bodies are generally immunogold stains (Dvorak et al., 1988). A third granule absent. The large immature granules in eosinophilic population. called small granules, contains hydrolytic myelocytes are precursors to primary and secondary enzymes (reviewed in Dvorak et al., 1991 a); these are granule populations. Late myelocytes contain mostly the small. dense structures of varying shape. latter. These immature granules are perfectly round and homogeneously dense. Dense, central crystalline cores 3. Immature human eosinophilic myelocytes with irregular shapes are present in small numbers in the maturing secondary granules. Earlier myelocytes have Eosinophilic myelocytes (Fig. 3) are immature larger numbers of it-nmature primary granules. These eosinophils. They generally are -3x larger than mature structures have loosely arranged. moderately dense, eosinophils and characteristically have an ample finely granular contents and are at times circumscribed cytoplasmic area filled with large, round immature by an outer layer of small vesicles beneath granule granules, extensive dilated cisterns of rough endo- membranes. Myelocytes with mixtures of these plasmic rericulurn and an enlarged Golgi complex. Their immature granules tlo exist. When crystalline cores surfaces have blunt cytoplasmic processes; the nuclei are appear in immature granules, however, they are Iarre. tnonolobed. lobular structures with partially second:tr!l granule precursors.

Fig. 2. The cytoplasm of a mature eosinophil (peripheral blood sample) shows secretory granules. Specific (secondary) granules contain dense cores filled with major basic protein (MEP), surrounded by less dense matrix where eosinophil- derived neurotoxin (EDN), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) (reviewed in Dvorak et al.. 1991a), and turnor necrosis faclor- n (TNF-a) (Beil et al., 1993) reside. Primary granules, the location of Charcot-Leyden crystal protein (CLC-P), are homogeneous and do not contain central crystalline cores (Dvorak et al., 1988) (with permission. Dvorak. 1991b). X 29,000 Morphology of human eosinophils in vitro

4. Tissue eosinophils granules). lipid bodies and tubulovesicles (Dvol-ak et al.. 199 1 a). Many of the morphologic changes that have Tissue eosinophils (Figs. 4-7) display many ultra- been recorded in tissue eosinophils also occur in factor- structural morphologic changes in a wide variety of supplernerited cultures of mature human eosinophils diseases (Dvorak, 1980; Dvorak et al., 1980, 1982, (Dvorak et al., 199 1 b, 1992). 1990a.b. 1993d; and reviewed in Dvorak. 1988; Dvorak Tissue eosinophil granule changes include decreased et al.. 199 1 a). Subcellular features include both numbers of specific granules (Fig. 6). looses from one quantitative and qualitative alterations in granules. or both compartments of these bicompartmentnl tubulovesicles and lipid bodies. The general processes in granules with retention of granule membranes, a process which tissue eosinophils participate include activation, termed piecemeal degranulation (PMD) (Fig. 5), as secretion and cell injury. Ultrastructural changes in cells well as increased numbers of primary (Fig. 5)and small undergoing secretion include those analogous to granules (Fig. 5). tubulovesicles and lipid bodies regulated exocytosis (Fig. 4) (Dvorak et al.. 1993d) and (Dvorak et al., 199 1 a). Lipid bodies may also increase piecemeal degranulation of contents considerably in size and display focal lucencies (Fig. 5) (Dvorak, 1980; Dvorak et al.. l99 1 b, 1992). and punctate densities in their interiors (Dvorak et Morphologically, cell alterations induced by in.jury to al., 1991 a). Tissue eosinophils have been shown eosinophils can be categorized as apoptosis. necrosis, to extrude membrane-free secondary granules into and mixtures of these processes (Dvorak et al., 1982, large, internal degranulation chambers or into the 1990b: Weller and Dvorak, 1994; Weller et al., 1994). extracellular milieu through multiple pores in the Cell activation changes include those of increased circumferential cell surface (Fig. 4) (Dvorak ct al.. granule populations (Fig. 5) (other than secondary 1993d). Cell injury of eosinophils with the characteristic

Fig. 3. An eosinophilic myelocyte (bone marrow sample) shows a single, eccentrically located lobular nucleus, extensive dilated cislerns of rough endoplasmic reticulum (RER), a Golgi structure, which contains a small progranule, and numerous large, dense, round immature secondary granules. Several immature granules, with finely granular material and surrounding electron-lucent space, are immature primary granules (with permission, Dvorak, J. Electron Microsc. Tech. 6, 255- 301. 1987).X 11.500 Morphology of human eosinophils in vitro

Fig. 4. A tissue eosinophil (Staphylococcal culture- positive biopsy of a continent pouch constructed from small bowel in an ulcerative colitis patient) shows focal extrusion of four secretory granules (open arrowhead). The granule cores retain their shape, and the matrix material has extended into a linear conliguration adjacent to the cores. All are contained ajdacent to a single surface pocket of the eosinophil. The remaining cytoplasmic granules show little evidence of morphological change. They are a mixture of secondary, primary and small granules. A single lipid body contains a narrow osmiophilic rim and multiple membranous structures (arrow). Note the collection of swollen enteric nerves with a few dense core granules nearby (closed arrowhead) (with permission, Dvorak et al.. 1993d). X 16.000 Fig. 5. The cytoplasm of a tissue eosinophil (skin b~opsyof a patient w~ththe hypereosinophilic syndrome (HES) and a pruritic skin rash) shows piecemeal degranulation (PMD) characterized by focal losses from the electron-dense cores of secondary granules (arrowhead), ~ncreased numbers of primary aranules lacking cores (closed arrow), and small granules (open arrow). Also, note the large number of cytoplasmic smooth membrane-bound vesicles and elongated tubules surrounding the cytoplasmic granules. X 41.000

Fig. 6. A tissue eosinophil (Staphylococcal culture- negative biopsy of an ileostomy from a patient with Crohn's disease; damaged axons were present in the same biopsy (Dvorak et al.. 1993d)) shows decreased numbers of secretory granules Several primary granules and one lobe of a typical eosinophil nucleus with condensed chromatin identify thls cell as of the eosinophil lineage. X 15.000 Morphology of human eosinophils in vitro

features of apoptosis or necrosis ultimately releases 5. Human eosinophils in vitro ~nenibrune-boundspecific granules into the tissue ~nicroenvironment(Dvorak et al.. 1982. 1990b; Ultrnstrucrural analysis of a large nurnber of cultures Weller and Dvorak, 1994, Weller et al., 1994). of human ~lrnbilicalcord blood cells, supplen~entedwith Necrosis of eosinophils is characterized by cell and a nurnber of growth factor-containing substances. has organellar swelling. broken cell and organellar provided insight into the varied morphology of membranes. and nuclear chromatolysis (Fig. 7). eosinophil\ in vitro. In a large sense, these studies Apoptosis is characterized by the formrttion of recapitulate previously documented human eosinophil cytoplasmic apoptotic bodies and nuclear chromatin morphology in vivo. The growth factors and sources used condensation, pyknosis. and formation of nuclear for these culture studies include the following: apoptotic bodies. i. interleukin-2-clepleted culture supernatant from

Fig. 7. A tissue eosinophil (skin biopsy of rash in a patient with the hypereosinophilic syndrome) shows necrosis. Note dispersion and leakage of nuclear chromatin into the cytoplasm and extracellular space (arrows) (chromatolysis) as well as the broken nuclear. organellar and plasma membranes. Cyto- plasmic contents and organelles are roughly within former cytoplasmic domains. In other areas of this biopsy (data not shown), membrane-bound secondary granules with focal core compartment losses were widely dispersed in the extracellular matrix. X 19.000 Morphology of human eosinophils in vitro

phytohemagglutinin (PHA)-stimulated human T iv. recombinant human interleukin-5 (rhlL-5) (Saito et lyrnphocytes, known to contain IL-3 - a hurnan al.. 1988; Dvorak et al.. 1989); basophilopoietin (Dvorak et al., 1985, 1993f): v. rhIL-5 and the culture supernatant described in (i) ii. murine T lymphocyte conclitioned media, known to above (Dvorak et al.. 199 1 b, 1992); contain IL-3 - a murine mast cell growth factor vi, fibroblast culture supernatants (Mits~~iet al.. 1993: (Dvorak et al., 1985, 1993f); Dvorak et al., 1994a); iii. recornbinant human interleukin-3 (rhIL-3) (Saito et vii. partially purified, naturally occurring mouse c-kit al.. 1988: Dvorak et al., 1989); ligand in 3T3 fibroblast culture supernatants (Mitsui

Fig. 8. An eosinophilic myelocyte (umbilical cord blood cell (UCBC) suspension culture with rhlL-3 for three weeks) shows eccentric, monolobed lobular nucleus w~thdispersed chromatin and large nucleolus, expanded Golgi structures, extens~vearrays of dilated cisterns of RER, elongated m~tochondna, and round, dense Immature secondary granules. Note the formation of irregular condensations of central dense core material (arrows) in the latter. X 16,000 Morphology of human eosinophils in vitro

et al., 1993: Dvorak et al., 1993b,c,e, 1994b); (rhGMCSF) (Weller et al., 1994). viii. recombinant mouse c-kit ligand (Mitusi et al., It is now known that additives containing human IL-5 1993; Dvorak et al., 1993b): best induce the development of the eosinophil lineage ix. reco~iibinanthuman stem cell factor (rhSCF - also from human umbilical cord cells in suspension cultures known as reconibinant human c-kit ligand and (Saito et al.. 1988: Dvorak et al., 1989, 1991b. 1992). recombinant human mast cell growth factor) (Mitsui However. variable numbers of eosinophils are et al.. 1993; Dvorak et al.. 1993b,c,e, 1994b); identifiable by electron rnicroscopy in each of the X. recombinant Iiunian gr'ani~locyte-sti11iuIiitingfactor suppleniented cilltures of human cord blood cells listed

Fig. 9. An eosinophilic myelocyte (UCBC supension culture with rhlL-5 for three weeks) shows a nucleus, cytoplasmic organellar content and immature granules that are similar to those of eosinophilic rnyelocytes arising in rhlL-3-containing cultures of UCBC (see Fig. 8) (with permission, Dvorak et al., 1989). X 16.000 Morphology of human eosinophils in vitro

above. This morphological primer for eosinophils in vitro lobed nuclei and condensed chromatin developed in is based on these studies. We present images in a these cultures (Dvorak et al., 1985). The secondary chronological sequence that parallels the course of granules contained eosinophil peroxidase in the completion of our studies. matrix compartment (Dvorak et al., 1985). Mature basophils far exceeded the number of eosinophils 5. 1. Eosinophils develop in cultures supplemented with in these cultures, however (Dvorak et al., 1985: naturally occurring human /L-3 in stimulated lymphocyte Ishizaka et al., 1985). Eosinophilic myelocytes were also supernatants noted in these cultures. They displayetl EPO in the perinuclear and rough endoplasmic reticular cisternae. Variable numbers of mature eosinophils with poly- Golgi structures and immature granules (Dvorak et al.,

Fig. 10. Eosinophilic myelocytos (UCBC suspension culture with rhlL-5 for three weeks, prepared with a cytochemical method to demonstrate endogenous peroxidase) show EPO in the perinuclear cisterna, RER cisternae. Golgi structures, the matrix compartment of secondary granules, and in immature granules (with permission, Dvorak et al., 1989). X 9.500 Morphology of human eosinophils in vitro

cells often had released EPO bound to them, bur the remaining specific gran~llesand synthetic organelles did 5. 2. Murine T lymphocyte-derived conditioned medium not contain residual EPO. Thus, the morphological (known to contain murine /L-3) stimulates the criteria for PMD were established in eosinophilic development of eosinophilic myelocytes but not myelocytes, which did not complete their miituration to mature eosinophils. The eosinophilic myelocytes that mature cells (Dvorak et al., 1993f). develop show apoptosis, necrosis and piecemeal degranulation 5. 3. Recombinant human /L-5 (or /L-3) stimulates the development in vitro of human eosinophils It was determined, by ultrastructural analysis of the eosinophilic myelocytes derived fro111 these cultures. By three weeks in culture with either of these that several processes were in operation. The recombinant human interleukins, eosinophilic myelo- ~norphologicalcriteria of apoptosis, necrosis and cytes were the predominant cells that developed (Figs. 8. mixtures of these illjury patterns were evident (Dvorak et 9) (Saito et al., 1988). These young eosinophils were al., 1985). Subsequent release of membrane-bound filled with peroxidase-positive synthetic organelles and granilles. cellular and nuclear debris. and apoptotic immature secretory granules and did not show any bodies resulted in avid phagocytosis by macrophages morphologic signs of apoptosis or necrosis (Fig. 10). IL- (Dvorak et al.. 1985). Soluble EPO was endocytosed by 3 also induced the formation of large numbers of viable, basophils ancl was stored in their granules (Dvorak et al., peroxiclase-negative basophilic leukocytes (Dvorak et 1985). Morphologically, sotile eosinophilic myelocytes al., 1985: Saito et al., 1988). Extending the interval of were not clamaged. These cells showed vesicular cultures, which were supplemented with either of these transport of EPO from specific granule matrix recornbinant human interleukins, to five weeks resulted compartments. The si~rfacesof these actively releasing in the maturation of eosinophilic myelocytes to mature

Fig. 11. A malure eosinophil (UCBC suspension culture with rhlL-5 for live weeks) shows a polylobed nucleus with condensed chromatin as well as core-conta~ninasecondary granules (closed arrow). The Golgi area sirrounds a centriole (open arrow) (with permission. Dvorak et al.. 1991a). X 15,000 (Bar = l pm). .i* .>v..'.. .- Morphology of human eosinophils in vitro

Fig. 12. Portions of the cytoplasm of mature eosinoph~ls(UCBC suspension culture w~thrhlL-5 for five weeks) show secondary granules at high magnification. Note irregular, thick, dense aggregates of core material (a-c) and mature secondary granule with dense core and less dense matrix compartments (d) (a, b. d with permission. Dvorak et al., 1989; c: with permlsslon. Dvorak et al., 1991a). (a) X 75,000; (b) X 81.000: (c) X 79,000: (d) X 51.500 (Bars: a-c = 0.1 pm, d = 0.2 um). 35 1 Morphology of human eosinophils in vitro

eosinophils with polylobed nuclei containing condensed was evident in these cultures (Figs. 14, 15) (Dvorak et chromatin (Fig. I I) (Dvomk et al., 1989). Basophilic al.. I99lb. 1992). This process was associated with myelocytes also ~~nderwentmaturation (Dvornk et al.. prominent collections of peri- and intragranular vesicles: 1989). losses were sustained froni both matrix and core The ~natul-ationof eosinophilic niyelocytes was compartments, giving rise to cmpty granule containers in accompanied by size reduction, elimination of synthetic the cytoplasm (Fig. 14) (Dvorak et al., 199lb). Other- organelles. chromatin condensation. segmentation of ~norphologicalaspects associated with activation. nuclei and granulogenesis. Maturation of the crystalloid- analogous to those seen in tissue eosinophils, were often containing specific granules was accompanied by the also present. The vesicular transport of specific granule formation of irregular, rope-like material and large matrix peroxiclnse was de~nonstratedin cytoche~nical irregular blocks of dense core material (Fig. 12). preparations (Fig. 15). TIILIS,vesicular transport of this Condensation of these patterns yielded central cores that secondary granule nlatrix protein was demonstrated were often irregularly shaped (Fig. 12). We noted losses to be an effector mechanism for PMD of mature from the matrix compartment of these granules. detected eosinophils in vitro (Dvorak et al., 1992). The cyto- by the electron lucency of this compartment. Some chemical preparations showed EPO in small vesicles that mature eosinophils had decreased numbers of specific were attached to specific granules, adjacent to granules. granules and increased numbers of tubulovesic~~lar in the peripheral cytoplasm and closely docked and structures. lipid bodies, primary and small granules. fused to the plasr-na nlernbrane. Secreted EPO was analogous to the changes seen in tissue eosinophils in attached to the cell surface in focal patches. The vivo (Fig. 13). peroxidase content of the matrix compartment of specific granules was either unaltered or partially 01- 5. 4. Recombinant human /L-5- and /L-2-depleted, PHA- completely absent (Dvorak et al., 1992). In contrast to stimulated human lymphocyte culture supernatant the transport and secretion of peroxidase-loaded vesicles stimulates the development of, and PMD from, mature from the specific granule matrix compartment, Charcot- human eosinophils Leyden crystal protein (generally stored in primary granules in peripheral blood eosinophils in vivo) filled PMD of the specific granules in mature eosinophils the cytoplasm LIP to, but not beyond, the subplasma

Fig. 13. A mature eosinophil (UCBC suspension culture with rhlL-5 for flve weeks) shows the virtual absence of all secondary granules. One core-containing secondary granule with matrix depletion remains (closed arrowhead) Lipid bodies (open arrowheads) and cytoplasmic tubuloves~clesare increased. Large, Irregular cytoplasmic electron-lucent spaces are glycogen aggregates that are not imaged with the method used here (with permlsslon, Dvorak et al., 1991a). X 13,000 Fig. 14. Mature eosinophlls (UCBC suspension cultures with rhlL-5 and IL-2- depleted, PHA- stimulated human lymphocyte culture supernatant for five weeks) show PMD. In (a), note the polylobed, mature nucleus and numerous enlarged. nonfused, empty and partially empty secondary granule chambers. Partially empty granule containers retain some dense granule material and vesicles. The remainder of the cytoplasm contains perigranular vesicles. mitochondria, small granules and a few strands of RER. In (b), at higher magnification the cytoplasm shows clusters of pengranular and intraganular smooth vesicles, some of which are electron- lucent (arrows) or contain lightly dense (closed arrowhead) or very dense contents (open arrowhead). Residual dense granule material is present in some of the granule chambers (with permission. Dvorak et al., 1991b). (a) X 15.500: (b) X 31.500 Morphology of human eosinophils in vitro

Fig. 15. Mature eosinophils (UCBC suspension cultures with rhlL-5 and IL-2-depleted, PHA-stimulated human lymphocyte culture supernatant for live weeks, prepared with a combined cytochemical technique to demonstrate endogeno~~speroxidase (a-c) and immunogold method to detect CLC-P (b.c)) show vesicular transport of EPO (a-c) but not CLC-P (b.c) in PMD. In (a), a control non-immune immunoglobulin was si~bstitutedfor the specilic acti-CLC-P primary antibody in the immunogold method. Note a single gold particle in the cytoplasm with none in the nucleus or over the Epon background. indicating exquisite specificily of the cytoplasmic gold-labeled CLC-P in (b.c). Numerous cytoplasmic vesicles contain EPO (arrows), which is of similar density to the EPO in the matrix of secondary granules. Focal areas in several peroxidase-negative secondary granule cores are permeated by EPO (compare morphology to that of Fig. 12a). EPO released from the cells is attached to the cell surface. In (b,c), secondary granules with EPO in their matrix compartments as well as EPO-replete vesicles, either adjacent (b) or attached (c) to EPO-containing granules (arrows), are present. Diffuse gold particles in the cytoplasm represent CLC-P (with permission. Dvorak et al., 1992). (a) X 34.000: (b) X 26,000: (c) X 33,500 Fig. 16. Mature eoslnophils (UCBC suspension culture wlth rhSCF at six weeks (compare with Flg. 1, small intestine biopsy, and Fig. 11, rhlL-5 containing culture of UCBC)) show polylobed nuclei with condensed chromatin patterns and many secondary granules. Their cell surfaces have Irregular, broad processes. The central core compartment of the secondary granules contalns irregularly shaped dense core material whlch 1s eccentrically located in some granules. In many secondary granules, a well-defined core compartment is not delineated. (a) X 16.500, (b) X 19,000 Fig. 17. An eosinophilic myelocyte (UCBC suspension culture with 3T3 fibroblast culture supernatan! for three weeks) shows an eccentric, single lobular nucleus with partially dispersed chromatin and a large nucleolus, dilated cisterns of RER. and numerous immature granules. Some of these contain small vesicles and irregular dense membranes. X 16.000

Fig. 18. An eosinophiltc myelocyte (UCBC suspension culture with rhSCF for three weeks) displays features similar to those seen in Figs. 3 (bone marrow sample). 8 (rhlL-3-), 9 (rhlL-5-), and 17 (3T3 fibroblast supernatant-containing cultures of UCBC). Note the condensation of dense core material in several immature secondary granules (arrows). X 14,500 a~o:, a6lel ha^ pue llews 'le~lua3 MO+ 'JaAaMOq 'IeJa~as'saln~el6 asaql jo lsow U! alq!s!h Al~ood ale sal03 lellua:, leql aloN .salnue~6 hepuo3as alnlewur! 'punoJ 'a6lel snolatunu pue 'Apoq p!d!~ auo 'saqol leapnu jo uo!leledas smoys (syaa~ 'A~SUJ aJnlln3 uo!suadsns m3n)alA301aAw 3111ydou!soeale1 e 10 tuseldolA:, aqi jo uo~l~odV '61 '6!j

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.punoj a.lam sa!8oloqd~ourSLI!MO~~O~ '(2661 "[c la yc.~o~a) aql LII!,M sl!qdou!soa alqn!j!~uap!/Cllcs!Soloqd~our (3'q~l'9!3) suo!lt?~eda~dlu.rri~sn~lse.~~ln ploflounmiu! *sa.rnl[nn asaq) do 11n 111 '(q'e~661'a'3Gq~3j[ "]c Aq pau!ulJalap SI? 's[[a3 i~1o.gpas1;ala.r iCla~!suaixaaq )a yn.roAa :~3j1"[c la !nsi!w) (+J~~~L~I.I)~013~~ q1~0.18 01 ~~!addn1011 p!p puc sllao palc~!lscasay] u! auwqiuaiu Morphology of human eosinophils in vitro

Fig. 20. Eosinophils and mast cells (UCBC suspension cultures with rhSCF, six weeks, prepared either with a routine ultrastructural method (a) or with a cytochemical method to detect peroxidase (b)) show ultrastructural and cytochemical differences which are characteristic of these two cell types. In (a), the mast cell (MC) is covered with narrow surface folds and contains a mixture of mature dense granules and immature. vesicle-packed granules. The eosinophil (E) shows secondary granules, many of which retain irregular cores and display expanded, empty (electron-lucent) matrix compartments. In (b), the eosinophil (E) shows EPO in

bodies also show peroxidatic activity (arrow). The mast cell (MC) reveals an absence of peroxidatic activity in granules; peroxidatic lipid bodies (arrow) are present. X 8,500 Morphology of human eosinophils in vitro

b. Eosinophilic myelocytes (Figs. 17- 19). distended cisternae. as well as expanded Golgi structures. Their cytopl~is~nis filled with larye. dense Eosinophilic myelocytes are also present in in immature granules, some of which display centrally vitro samples. These immature eosinophils (Figs. 17- condensing crystalline cores. Some irnniature granules 19) are approxilnately three-times larger than mature contain less dense materials. often surroundetl by eosinophils. Their nuclei are not polylobed; rather. small intragranular vesicles. Intragranular. dense large, lobular, monolobed nuclei with dispersed ~nembratiousstructures can also be found. Recognizing chromatin and large n~~cleolicharacterize eosinophilic these subcellular features will prevent confusion myelocytes. The surface architecture consists of with i~nmaturehuman mast cells, which are regularly irregular, broad processes. Eosinophilic myelocytes have present in the same culture samples (Fig. 20). The irse large arnoilnts of rough endoplnsniic reticulum with of naturally occurring c-kit ligand, which is present

Fig. 21. An eosinophilic myelocyte (UCBC suspension culture with rhSCF, three weeks) shows extrusion of nonmembrane-bound, dense primary granules (arrowheads) through the plasma membrane. X 17,500 Morphology of human eosinophils in vitro

Fig. 22. Eosinophilic myelocytes (UCBC suspension cultures with fibroblast culture supernatant (a) or rhSCF (b), three weeks, prepared either by a routine ultrastructural (a) or by a cytochemical method to demonstrate endogenous peroxidase (b)) show secretion of primary granules by extrusion (arrowheads). In (a), a single granule is being released through the plasma membrane; in (b), numerous extruded, peroxidase-containing granules remain adjacent to the cell surface. The perinuclear cistern and RER cisternae contain EPO in (b) (N = nucleus). (a) X 25.000; (b) X 35.000 Fig. 23. Mature eosinophils (UCBC suspension cultures with rhSCF, six weeks) show PMD, characterized by expanded, empty, electron- lucent matrix compartments of secondary granules. Several unaltered, bicompartmental secondary granules remain (arrows). Residual, irregular dense core material is present in some granules (arrow- heads). (a) X 16,000; (b) X 13.000 Morphology of human eosinophils in vitro

in fibsoblast supernatants, or the use of rhSCF, as d. Morphological evidence of regulated secretion a hurnan cord blood cell culture additive, resulted from eosinophilic rnyelocytes which develop in vitro in the developlnent of eosinophilic lnyelocytes (Figs. 2 1. 22). and their mature progeny. which did nor differ morphologically. Reg~~latedsecretion from secretory cells is defined niorphologically by the extrusion of membrane-free c. Members of the eosinophil lineage differ from granules from granulated secretory cells. We previously immature mast cells. described this process in vivo in bone marrow eosino- philic lnyelocytes (Dvorak et al., 1991a) and in mature Eosinophils can be distinguished frorn immature tissue eosinophils (Dvorak et al., 1993d). Eosinophilic mast cells. which regularly develop from suspension myelocytes developing in vitro also have the capacity for cultur.es of human cord blood cells supplemented regulated secretion. For example, and as in bone marrow with the c-kit ligand (Fig. 20). In routine electron in vivo. eosinophilic nlyelocytes extrude imlnatul-e ~nicroscopicpreparations, the surface architecture primary granules, which are ~noderatelydense, finely and the immature granules of developing mast granular. nonmembrane-bound structures devoid of cells clearly differ from those in eosinophils (Fig. crystalline cores. through pores in the plasma membrane 20). mast cells. for example. are characterized (Figs. 2 1. 22). This secretory mechanisni of irnrnature by narrow surface folds and large numbers of primary granules Inay account for the small numbers variably dense i~urnaturegranules. Ultrastructural (-59) of retained primary granules that are seen in cytochernical preparations to detect endogenous mature circulating eosinophils (Dvorak et al., 1988). The peroxidase show peroxidase only in immature primary granule is the only eosinophil granule storage mast cell; eosinophil granules, lipid bodies and site of Charcot-Leyden crystal protein, as determined synthetic organelles stain positively with this techniclue by immunogold ultrastructural studies (Dvorak et al.. (Fig. 20). 1988).

Fig. 24. A mature eosinophil (UCBC suspension culture with rhSCF, six weeks) undergoing PMD shows clusters of electron-lucent vesicles (arrowhead) adjacent to an enlarged secondary granule which has residual core material and an empty matrix compartment containing several vesicles. X 17.500 Morphology of human eosinophils in vitro

e. PMD ofeosinophils in vitro (Figs. 33-36). effected PMD in mature and immature eosinophils in vitro (Dvorak et al., 1992, 1993tl. Mature eosinophils. PMD is a term we chose to define o secretory process developing in c-kit ligand-supplemented cul~uresof in grani~latedsecretory cells that differs rnol~phologically human cord blood cells, fulfil the ultraslructural from the regulated extrusion of entire ~ranulesfrom morphological criteria for PMD (Fiss. 23-36). Thuh. secretory cells (Dvorak, 1991~1,1993). In PMD, for variable numbers of secontlary granules with empty. full. example. granule contents are released in the absence of and partially empty granule containers remain in the granule-to-granule or granule-to-plasn~amembrane cytoplasm. Some secondary granules with dense inner I:iisions. with subsequent retention of granule containers cores and less dense matrix compartments are in the cytoplasm. Furthermore. we used a cytochernical unchanged (Fig. 23a), whereas other secondary granules method to demonstrate that EPO-loaded vesicles are enlarged and partially or completely empty (Fig.

Fig. 25. A mature eosinophil (UCBC suspension culture with rhSCF, six weeks) is undergoing extensive PMD of -50?6 of the secondary granules. Note the clustered, electron-lucent smooth vesicles near releasing granules (arrowheads). Full. dense vesicles of similar size are also present near and attached to (arrows) secondary granules. X 14.000 Morphology of human eosinophils in vitro

Fig. 26. Mature eosinophils (UCBC suspension cultures with rhSCF for three weeks (a) or six weeks (b)), undergoing extensive PMD, show release from virtually all secondary granules. Their expanded containers remain in the cytoplasm and are surrounded by smooth vesicles. Irregularly shapeddensecore material (arrow) remains in some of the enlarged. electron-lucent granule chambers. (a) X 12,000; (b) X 15,000 Fig. 27. An eosinophil (UCBC suspension culture with rhSCF, six weeks) shows apoptosis. characterized by a pyknotic nucleus filled with a crescent of dense chromatin. Note the absence of chromatin leakage and the absence of damaged cellular membranes, in contrast to what is seen in necrosis in vivo (compare with Fig. 7). X 13,000

Fig. 28. A mature eosinophil (UCBC suspension culture with rhSCF, six weeks) shows increased, abnormally shaped aggregates of core material in two remaining secondary granules (arrowheads) and focal release of the dense core from another (arrow). Increased numbers of small granules and tubuloves~cularstructures are present. X 17,000 365 Morphology of human eosinophils in vitro

23b). Retention of core material, despite completely Thus. in addition to neutrophils (Dvorak et al.. 1993e). empty matrix compartments, was regi~larlyobserved some eosinophils morphologically expressed apoptosis (Fig. 23b): focal losses frorn core compartments of (Fig. 27). These cells were characterized by extensive secondary grani~leswith retention of matrix contents. as condensation of nuclear chromatin. Chromatolysis of regularly occurs in tissue eosinophils from some nuclei and lysis of membranes - feati~resof necrosis - diseased tissues in vivo, were not regular features of were not prominent in these cultures. Other lineapes PMD that we observed in vitro. Clusters of electron- simultaneously present (viz., mast cells. basophils. lucent ancl electron-dense vesicles adjacent to secondary macrophages. endothelial cells) did not show apoptosis granules were regular features of PMD in vitro (Figs. 24- or necrosis. 26). Some of these vesicles were fused to granules. Some eosinophils undergoing extensive PMD could be g. Qualitative morphologic changes in the secondary definitively recognized by the retention of irregularly granules of mature eosinophils in vitro (Figs. 28-3 1 ). shaped core material within electron-lucent secondary granules (Fig. 26b). Sorne secondary granules in mature eosinophils revealed abnortnally increased amounts of dense core f. Apoptosis material (Fig. 28). This material was arranged as large, irregular blocks and intertwined threads, as we noted in Apoptosis (programmed cell death) was evident in rhIL-3- or IL-5- supplemented cultirres (Dvorak et two cell lineages developing in c-kit ligand- al.. 1989). Rarely, we also found focal core losses in supplemented cultures of human umbilical cord cells. these granules (Figs. 28, 29). At times, the matrix

Fig. 29. A mature eosinophil (UCBC suspension culture with rhSCF, six weeks) shows diminished numbers of secondary granules and increased small granules. One secondary granule (arrowhead) has released its dense core. and the matrix compartment contains dense contents and numerous vesicles. X 16.500 Morphology of human eosinophils in vifro

Fig. 30. Mature eosinophils (UCBC suspension cultures with rhSCF. six weeks) show markedly enlarged secondary granules (arrowheads). In (a), all secondary granules are enlarged, and many have irregular collections of dense core material. Several osmiophilic lipid bodies (arrow). surrounded by lubules of smooth endoplasmic reticulum (SER), are present. In (b) secondary granules have electron-dense protrusions, suggesting electron- dense vesicle attachments (arrow). (a) X 20.500; (b) X 14,000 OOO'CLX (4) :OOS'PLX (P) .0d3 u!eluo3 salflUeJ6 AJepU03as Jalleus pue a(nue~6lue16 aql '(q) U! :sap!saA pue salnue~basuap 'llews Aq papunoJJns S! alnue~61ue16 ay1 '(e) ul 3alnue~6 1ue16MO~S ((q) ~SEP!XOJ~~snoua60pua walap 01 auo le3!waq301A3 e JO (e) poylaw le~nl3n~lse~lln au!gnoJ E ~aq]!a411~ pa~eda~d 'syaaM XIS 'j~sy~ ql!~saJnlln3 uo!suadsns 393n) sl!qdou!soa aJnlew 10 wseldolh ay1 jo suo!l~od'CC '6!j

~LII: itrn[113!1a~s!urs~!ldopua q~oc)iirs3!~11~1?ld011(3 jo slunoiut: ad.1~1jo a3uasa.rd ~LIIapnlxq .sl!qdou!soa sc slla:, .1ca13n~rLlod'~ulnu~?.rS!nn~?d ;i'ir!z!uSona.r u! [njdlay a.lc qs!q~'spclap ~cuo!l!ppl: a[uos '(2c-j-j1 '.IC la yc.ro~a)iira~ildola~ap jo aacls alA3olaA~u ~.\!IJLI!IS!P ay1 puoLaq aJnleru IOU p!p sl!qdo.r~nau pill? '(Jc(,~L '(q I %A) sa11ii1~14L~npuosas paz!s-(cu~.rou ay] put? "11' la yl?Jo,ia) awd jo suS!s paLt:lds!p /Cllo.~auaa a8~claqr q~oqU! a~uiCzuasriouaSopua palcaAs.1 O~ZJjo sl!qdoseq 'snqL '(a'3~661"[c 1a yc.loAa) sa.rnlln:, ~cl!u!s uo!lc.rlsuoulap aql ~ojsuo!~e.~eda~d ~~?s!uraqno~A~ alc!~tl U! A[lc3!1curc~ppaJajj!p sa4cau!l a~Laol~~cir.r;i'~r?uo!l!ppc -o~ddv'(I:[ c '3!3) sarnl3nJls punoq-anu.rqu~au~.asuap OM] asail] jo .rno!ncqaq ay] :iuasqc aJaM 'sl!ildo.rlnau 'IIPLIIS sncxraurnu Lq papuno.r.rns pire -xuap-uo.r~nala puc sl!qdosuq jo ~!Is!.I~I~I:.I~~I~'salnu1:~8 iC.rola.rnas L[alalduros 'puno.~aJaM sa[nuc.rZ A~cpuo~aspaS.rc[ua .sassa3o.rd anl?.l.rns .rc[nSar.r! .r!aql ;Cq poc u!lcuro.rqn ay] 'ualjo '( 1~ '(1s .~S!A)az!s [I:LUJ~U jo saln~rl?.lS pasuapuos ql!~!a[snu paqolAlod .[!ay] Sq salAnolnue.ri? ol LIO!I!~~I?U! salnumS A.rcpuooas paS.rclua Ic.ra.tas .ro auo SI? a[qc-/!uSosa.r a.laM slla3 asall1 '(cc .3!.~)luasa.rd Jaqi!a paq sl!qdou!soa a.lnn:iir .raqlo :(OC 'S!d) Il:!.ralciu oslc a.laM sa(1iirc.13L~t?pi~onas ;10 p!o,\ap Llaialdir~o:, a.103 jo saluSa.rXc asuap ..rcln8a.r.r! paAelds!p i[s!q,m a.ra,m II?~Isl!qdou!soa alnlciir auras .(q;c 'i?! J) y.rni111~q jo auros 'salnuu.12 ;C.rcpuonas pa3.1clua Aluo pau~cliron Su!L~!~uap!J!aql S! salnrrc.13 hlcpiro3as ;?'u!u!orua.r aql do sI!ildou!soa a.rnlt:iu amos '((jZ .S!g) ~uauri.rcdiu(~~ss!ql s.roiu .IO a~loLI! IC!.I~II?~U a103 astrap jo iro!lt:.~.rasa.~dailL jo s~uauodii~osasuap aql II!~I!M sal:,!sa,i iua~n[-uo~l3ala '(cc 'zc .S~!.J)oqn U! palou aJsM salriir~:.rS,C.~t:puosas ql!~173111~ SI?M salliirc.13 L.r~pi~onasso l~raii~~.r~?diiro:, Fig. 32. Mature eosinophils (UCBC suspension cultures with rhSCF, six weeks) show diminished numbers of secondary granules. In (a),the granule-poor cytoplasm is filled with SER and lubulovesicular structures. In (b), core material is preserved in one remaining granule (arrowhead). (a) X 14,500; (b) X 17.500 Fig. 33. Mature, granule-free eosinophils (UCBC suspension cultures with rhSCF, six weeks) are recognizable by their typical polylobed granulocyle nuclei. In (a), a single primary granule remains; the cytoplasm is filled with SER and tubulovesicular structures. In (b) two lipid bodies (arrowhead) are seen. (a,b). X 16.000 Morphology of human eosinophils in vitro

Fig. 34. Mature eosinophils (UCBC suspension cultures with rhSCF, eight weeks) show condensed. polylobed granulocyle nuclei. In (a), the eosinophil has large numbers of round, osmiophilic lipid bodies (arrowheads) and piecemeal losses from secondary granules. In (b), the eosinophil has two large lipid bodies (arrowheads). Nearly all remaining granules are homogeneously dense primary granules. Bicompartmental secondary granules are absent. (a) X 12.000; (b) X 17.500 00~'~1 X 'tuseldolk pue snapnu a41 UI luaso~d osle S! d-3-19 asnwa '(SMOJJE) salnuel6 /(J€?u!J~ P~I~CI~I-PIO~smovs (d -313 13alap 01 powaw p106 -ounuw! ue 411~pa~eda~d pue 'syaaM x!s '43~4~ UI!M a~n1ln3uo!suadsns 383n3 1ludou!so3.SE '6!d

.(I? 1 66 1 "[l? la y?.roAa a.rnisur ~cn!dL~qi!~ sl!qdou!soa a.rnlcur amos tr! pa,cl\a!,\a.r) eaurLzua n!~Llo.rpAt~u!l:itron oi u~ooy .Luaqi U!YI!Msaln!i.rcd asuap put? sca.rn iuasnl-uo.r~nala a.rt? icql sa.1ninn.11~- (c)~ 39) "lnirc~3 punoq-suc.rqura~u ~csojpaMoys iCircur pur! '(qpc '3!~)p".~~:lirs a.ram 'astrap '~lciirsjo s.taqurnu pasna.~nu!paL~?ldc!p salnu~?.rd aiuos :puno.r iC~~t?~auaSaJam sa!poq p!d!7 '(pc slla:, ,C.rl:puosas paqs!u!iu!p ill!i~SI!II~~U!~O~ .raqiO poolq nnurnq pa.rn11nn uro~jSu!s!.rt? sllan aJnicLu '(~c 0.y~U! sl!qdo~r!soa u! sallauc3.1o asaql jo sJaqiunu pacl?aJsu! paiou ua!lo a.+\ jo enalnnu put urscldoiAn aql U! iuasa.ld osln sl?,.~.(q 1661 'snyl 'O~AU! saZucqn a,\!icl!iut?nb pun a.\!ll!l!lnnb ;wqs "lr! ia yn.lo,~a)salnilno poolq p.103 ucurnq ~~a~us~irqldd~isicqi sa!poq p!d![ punoJ ':,!l!ydo!wso -punoq-aur?~qu~au~ -~-71il.rLI! Z~r!s!.rl? sl!ydou!soa a.rnjuur U! pa~c.lisuoiuap -uou jo s.raqLunu alqc!.tl?,t aAcq sl!qdou!eoa a.rnlcl,q ,(lsno!.\a.rd anuq s~ sr! 6u!aio~ds!qi .raj [aqcl p109 '(SS 'S!g) su!nls ploSoun~uur!In.InlnnJlsr!.llln ,Cq pa~c.rlsuourap st? 'u!aio.rd ~c~sL.rr,uapLal-ion~cy3 pau!cliro:, aJniln:, u! 3u!s!.rc sl!qdou!soa u! salnun.rJ ,C.rciu!.rd ~LIJ, '(qtc salnuc.13 IC.rnu~!.rd actlap ,(lrnoauaSou~oq jo s.raqurnu pascaJsir! pau!nluor, inq sslnua.12 ,C~l:pi~onax ~l?ii~a~~i.~t?d~iror,!qjo p!o,tap a.1a.U !alnnir ai,C:,ol~itrr!.~S

Morphology of human eosinophils in vitro

murine growth factors on human and eosinophil maturation. Dvorak A.M.. Estrella P., Mitsui H. and lshizaka T. (1993e). c-kit ligand Lab. Invest. 53, 57-71. induction of immature neulrophils in cultures of human umbilical cord Dvorak A.M., Letourneau L., Login G.R., Weller P.F. and Ackerman S.J. blood. Eur. J. Cell Biol. 62. 422-431. (1988). Ultrastructural localization of the Charcot-Leyden crystal Dvorak A.M.. Estrella P. and lshizaka T. (1993f). Vesicular transport of protein (lysophospholipase) to a distinct crystalloid-free granule peroxidase in human eosinophilic myelocytes. Clin. Exp. Allergy. (in population in mature human eosinophils. Blood 72, 150-158. press). Dvorak A.M.. Saito H., Estrella P,. Kissell S.. Arai N. and lshizaka T. Dvorak A.M., Mitsui H. and ishizaka T. (1994a). Stimulation of partial (1989). Ultrastructure of eosinophils and basophils stimulated to development of human mast cell progenitors by supernatant fluid develop in human cord blood mononuclear cell cultures containing from mouse libroblast cultures. Clin. Exp. Allergy (in press). recombinant human interleukin-5 or interleukin-3. Lab. Invest. 61, Dvorak A.M., lshizaka T., Letourneau L.. Albee E.A.. Mitsui H. and 116.132. Ackerman S.J. (1994b). Charcot-Leyden crystal protein distribution Dvorak A.M.. Letourneau L., Weller P.F. and Ackerman S.J. (1990a). in basophils and its absence in mast cells that differentiate from Ultrastructural localization of Charcot-Leyden crystal protein human umbilical cord blood precursor cells cultured in murine (lysophospholipase) to intracytoplasmic crystals in tumor cells of fibroblast culture supernatants or in recombinant human c-kil ligand. primary solid and papillary neoplasm of the pancreas. Lab. Invest. J. Histochem. Cytochem. 42. 251-263. 632, 608-615. Dvorak A.M.. Morgan E.S.. 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Structurally distinct, nonmembranous intracellular siles of eicosanoid Weller P.F.Marshall W.L.. Lucey D.R.. Rand T.H., Dvorak A.M. and formation. In: Proslaglandins, leukotrienes, lipoxins and PAF. Bailey Finberg R.W. (1994). Infection, apoptosis and killing 01 mature J.M. (ed). Plenum Press. New York. pp 353-362. human eosinophils by human immunodeficiency virus-l. (submitted).