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Genesis ultrastructure and cytochemical study of the cat eosinophil.

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THE ANATOMICAL RECORD 196:119-127 (1980)
Genesis, Ultrastructure and Cytochemical Study
of the Cat Eosinophil
B. PHESENTEY, 2. JERUSHALMY, M. BEN-BASSAT AND K. PERK
Rogof- Wellcome Medical Research fnstitu.te, Tei-Auiu Uniuersity Medical
School, J . Casper Department of Patholog,y, Reilinson Hospital, Petah Tikun,
Hematological Department, District Lahoratory, Kupath Holim, Rehouot, and
Department of Animal Anatomy and Ph,ysiulog,y, The Hehrew Uniuersity,
Rehouot, Campus, Israel
ABSTRACT
Eosinophils from cat bone marrow and peripheral blood were
studied by electron microscopy and cytochemical procedures. The maturation of
eosinophils and formation of typical granules were described. Contrary to the
accepted opinion that the core of animal's eosinophilic specific granules have a
crystal-like structure, our observations revealed that the core has a myelin-like
cylindrical appearance, whose layered formation proceeds from the inside outwards.
Electron microscopic observations revealed that localization of reaction product
to potassium pyroantimonate and phosphotungstic acid and to acid phosphatase
activity was similar to that of eosinophils of man and other animals. Antimonate
deposits and acid phosphatase activity were detected between the layers of the
myelin-like structure of the core. Eosinophil granules failed to yield a positive
reaction for peroxidase activity. The secretory activity of the eosinophil is discussed.
MATERIALS AND METHODS
Use of histochemical techniques together
with electron microscopy permits determination of the location of enzymes and other constituents within eosinophilic granules (Ackerman and Clark, '71; Ackerman, '72; Bainton
and Farquhar, '70; Geger et al., '70; Hardin and
Spicer, '71; Hardin and Spicer, '70; Kelenyi and
Zombai, '64). Tnese granules have been designated a s lysosomes (Baintonand Farquhar, '70;
Faller, '66; Fawcett, '69; Zucker-Franklin, '68;
Geyer et al., '70; Kelenyi, '68; Leder and Paper,
'71; Presentey, '69; Scott and Horn, '70),on the
basis of their high enzyme content, particularly
of acid hydrolases (Archer and Hirsch, '63;
Bainton and Farquhar, '70; Geyer et al., '70;
Presentey, '69). In eosinophils of different animals, the enzyme content of the granules varies
(Jain, '68; Nakamura, '55; Presentey, '781, as
does also the relative dimensions of the inner
and outer regions of the granule. Developing
and mature cat neutrophils were studied by
Ackerman ('68). The present study traces the
course of development in the cat eosinophil,
starting from the undifferentiated blast stage,
the ultrastructural features, and cytochemical
reactions.
000-3276x180/1962-0119$02.00 0 1980 ALAN R. LISS, INC
Five mature cats with eosinophilia in excess
of 15% were selected for this study. Blood samples were withdrawn into heparinized test
tubes and kept a t room temperature for 30 min;
the buffy coat was collected and processed according to Watanabe ('59). Aspirated bone marrow was processed according to Ackerman and
Clark ('71). To test the reaction to phosphotungstic acid IPTA) during dehydration, the
procedure of Hudson ('66) was used. Peroxidase
activity was detected using the technique of
Ackerman and Clark ('711, and acid phosphatase using that of Seeman and Palade ('67).
Reaction to pyroantimonate was detected in
nonglutaraldehyde-treated buffy coat or
bone-marrow according to the method of Hardin and Spicer ('71).
Samples were fixed for 1hr in 2.57~buffered
glutaraldehyde, postfixed for 1 hr in osmium,
dehydrated in graded ethanol, and embedded in
epon. Sections examined for enzymic activity
Received September 26, 1978; accepted July 3, 1979.
This work 1s part of Ph. D. thesis submitted by B. Presentey to the
Tel-Aviv University.
Reprint requests to B. Presentey, Head, District Laboratories,
Kupat Hohm, P 0. Box 1300, Rehovot, Israel.
119
120
B. PRESENTEY, Z. JERUSHALMY, M. BEN-BASSAT AND K. PERK
and thin sections treated with PTA or pyroantimonate were unstained. Ultrathin sections
were double stained with uranyl acetate and
lead citrate and examined with a SEM-7
electron microscope.
RESULTS
Electron microscopic observations
The blast cell stage was characterized by a
large nucleus occupying the major part of the
cell. The nucleolus was well defined; small
amounts of heterocromatin could be observed near the nuclear membrane. The cytoplasm was rich in endoplasmic reticulum and
occasionally displayed dilatations. Mitochondria and Golgi apparatus were also evident.
The transitional cells, the early promyelocytes (Fig. l), were characterized by their large
size and by a large nucleus with more heterochromatin than in the blast cell, possessing a
large nucleolus and several granules composed
of a homogeneous, electron-dense substance. In
some of the granules, a differentiation in two
parts was evident. Endoplasmic reticulum
were numerous and their cisternae were more
distended than in the blast cell; mitochondria
were also r a t h e r numerous (Fig. 2) . The
number of granules increased during maturation. Granules were composed of two parts: a
darker one-core-and
a lighter one-matrix
(Fig. 2). With cell maturation and transition to
myelocyte, the nucleus diminished; the nucleolus became for the most part undetectable and
the amount of heterochromatin increased.
There was less endoplasmic reticulum and
fewer mitochondria in mature cells as compared to more immature stages. However, the
number of granules increased as did the dimensions of their core (Fig. 3 ) .
The mature cat eosinophil was characterized
by a segmented nucleus, cytoplasm which
abounded in granules and contained very few
mitochondria, and by endoplasmic reticulum.
In the specific granules, matrix was very well
detected; the core had a layered myelin-like
structure. Occasionally, however, the granules
contained only a few myelin-like fragments
(Fig. 4). Some granules were lacking normal
core structure. The latter became lighter from
the matrix, giving the impression that its content had been released (Fig. 5 ) . In addition, the
mature eosinophil contained microgranules,
round or oval i n shape, and sometimes
double-walled (Fig. 5 ) , similar to those described in man (Parmley and Spicer, '74;
Nichols and Bainton, '73).
Reaction of eosinophils to phosphotungstic
acid IPTA)
While a positive reaction to FTA was usually
observed in the matrix of the granules, in a few
cases this reaction did not take place, though
the cells did not appear to have suffered any
damage (Fig. 6).
Reaction of eosinophils to potassium
pyro-antimonate IPPA)
A strong positive reaction to PPA occurred
mainly in the matrix of the granules, but positive reactions were also observed between the
layers of the inner, myelin-like part (Fig. 7).
Granules without any evidence of destructive
changes occasionally failed to react positively.
As in human eosinophils, there was a positive
response in the region of heterochromatin material in some of the nuclei.
Reaction of eosinophils to peroxidase
Cat eosinophils did not react positively for
peroxidase; this is in agreement with findings
using cytochemical procedures and light microscopy (Fig. 8).
Localization of acid phosphatase activity
in eosinophils
A strong positive reaction for acid phosphatase was observed in the matrix, in the layers of
the inner, myelin-like region, and in the center
of the eosinophil granules (Fig. 9). In some
cells, a positive reaction was observed in the
nucleus and the intergranular cytoplasm,
while the granules themselves displayed positive or negative reaction (Fig. 10).In other cells,
positive reactions occurred only in the intergranular cytoplasm and in the nucleus, in
which heterochromatin was not detected. No
destructive changes could be observed in the
nonreacting granules (Fig. 11).
DISCUSSION
Even in cases of pronounced eosinophilia, the
number of eosinophils in bone marrow remains
small. Consequently, investigators of cat eosinophils have so far limited themselves to a
thorough study only of the mature eosinophil
(Bargmann and Knopp, '58; Fawcett '69; Ward
et a1 '72). We studied the ultrastructure of the
bone-marrow eosinophils, including all stages
of blast maturation. The granules first appear
in the promyelocyte, and core and matrix are
formed. Simultaneously, the process of granule
GENESIS, ULTRASTRUCTURE OF CAT EOSINOPHIL
Fig. 1. An early promyelocyte-probably a n eosinophilic one. Large nucleus, delicately wrought, with heterochromatin a t the periphery and a prominent nucleolus. The cytoplasm contains many profiles of rough endoplasmic reticulum (some dilated), mitochondria, and granules of varying electron density. Uranyl acetate and lead
nitrate x 3,720.
Fig. 2. An immature eosinophil-a late promelocyte with large nucleus and nucleolus. Abundant endoplasmic
reticulum with expansions. Increase of specific granules. Core and matrix are identified in all of them. Uranyl
acetate and lead nitrate x 4,185.
121
122
B. PRESENTEY, 2. JERUSHALMY, M. BEN-BASSAT AND K. PERK
Fig. 3. Three eosinophils at different stages of maturation. One l:ite promyelocyte, one myelocyte, and one
methamyelocyte. In the more mature cells the nucleolus has disappeared, the nucleus became smaller, and the
hekrochromatin more abundant. Cytoplasm is filled up with typical granules. Uranyl acetate andlead nitrate x
2,325.
Fig. 4. Portion of mature eosinophil. Layered structure of myelin-like core of specific granules is discernihle
Uranyl acetate and lead nitrate x 37,200.
GENESIS, ULTKASTRUCTURE OF CAT EOSINOPHIL
123
Fig. 5. Portion of mature eosinophil. In three typical granules the core is lighter than the matrix (thick
arrows). The intergranular cytoplasm contains round or elongated microgranules, some of which are doublewalled (thin arrows). Uranyl acetate and lead nitrate x 18,600.
Fig. 6. Mature eosinophil following treatment with phosphotungstic acid during dehydration. Positive reaction isdiscernihle in the matrix of specific granules without additional staining. x 6,510.
124
B. PRESENTEY, 2. JERUSHALMY, M. BEN-BASSAT AND K. PERK
Fig. 7. Detail of mature eosinophil following treatment with potassium pyroantimonate. Positive reaction
discernible in outer part of typical granules and between the layers of the myelin-like inner part, without
additional staining. x 15,810.
Fig. 8. Bone marrow following incubation to detect peroxidase. Positive reaction in neutrophil, but not in
eosinophi. Uranyl acetate and lead nitrate X 3,720.
Fig. 9. Positive acid phosphatase reaction in matrix of specific granules. Positive reaction discernible between the layers
of the myelin-like structure of the inner part. Without additional staining. x 15,000. Inset x 50,000.
Fig. 10. Mature eosinophil following treatment with acid phosphatase. Positive reaction in some of the granules, but many
of the granules remain negative, although they show no visible degernerative changes. Positive reaction detectable in the
intergranular cytoplasm and in the heterochromatin of the nucleus. It is suggested that the enzyme evident a t these sites
originates from the granules, and that such granules, which have released their enzyme contents, are consequently rendered
negative to acid phosphatase. x 7,000.
Fig. 11. Mature eosinophil with a trilobate nucleus (N).Strong positive reaction to acid phosphatase in the intergranular
cytoplasm and in the nucleus, suggesting acid phosphatase release. Unlike Figure 10, no heterochromatin discernible in
nucleus. All the granules appear intact (arrows). x 6,000.
formation encompasses changes in both regions. Mature specific granules reveal a
myelin-like structure of the core.
The data presented indicate that it is not
feasible to attempt to distinguish different
categories of typical granules; contrary to the
cat, in human eosinophilic granules, the matrix
forms later than the core (Bessis and Thiery,
'57; Capone et al., '64; Fedorko, '68; Miller et al.,
'66; Scott and Horn, '70). Treatment with phosphotungstic acid a t times concealed the layer
structure of the inner part of the granules, resulting in a myelin-like appearance. Quintarelly et al. ('71) confirm that staining with
phosphotungstic acid is not specific for phospholipids (Presentey and Jerushalmy, '79;
Presentey, '78).
The response of the granules to potassium
pyroantimonate is similar to that already reported by other investigators of human eosinophils and of various laboratory animals (Ackerman, '72; Hardin and Spicer, '71; Hardin,
'70). The positive reaction observed between
the layers of the core a t its center suggests that
formation of this myelin-like structure proceeds gradually outwards. In addition to N a +
cations, potassium pyroantimonate precipitates also Ca+ and Mg cations, but this can
be partly suppressed under certain conditions
+
++
(Bulger, '69; Clark and Ackerman, '71; Klein et
al., '72).
Responses to acid phosphatase in the cat eosinophil follow a similar pattern to those found
in other animals and man (Geyer et al., '70;
Miller andHerzog, '69; Seeman and Palade, '67;
Wetzel et al., '67), but are stronger and in
agreement with findings by histochemical
techniques (Presentey, '78). In several instances a positive response was recorded in the
cytoplasm and nucleus, with some of t h e
granules responding positively and others
negatively (Fig. 10). We also observed cells
where all the granules failed to react, although
the intergranular cytoplasm and the nucleus
reacted positively (Fig. 11).These findings reveal t h a t t h e enzyme released from t h e
granules accumulates within the cytoplasm
and the nucleus, in the initial stage in the heterochromatin region and later in the entire nucleus causing damage to its structure. This observation confirms that intergranular response
to acid phosphatase is not an artifact, as it has
been previously interpreted (Seeman and
Palade, '67).
Secretory activity by eosinophils had been
postulated and was confirmed by Scott and
Horn ('701, who showed that release of entire
granules can be associated with release of the
GENESIS, ULTRASTRUCTURE OF CAT EOSINOPHIL
granule content-either of the core or of the
matrix. We found that negative responses by
granules was due to secretory processes (Figs.
1 0 , l l )and not t o degenerative changes, such as
described by Kelenyi ('68) and Kelenyi and
Nerneth ('72).
Basic proteins in the cat eosinophil have been
found to be the primary constituent of the core
of the granules and are not present in the outer
part (Lewis et al., '78), as claimed by Faller
('66).Contrary to Faller ('66),we found no evidence of the presence of various unsaturated
acids in the granules.
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Submitted
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