Ultrastructural features of eosinophil leucocytes of the goat

J co:w.

~"ATH.

1976.

\'OL.

183

86.

LiLTRASTRUCTURAL LEIJCOCYTES I.

GRANULES

FEATURES OF THE

DEVELOPMENT

IK

THE

OF EOSINOPHIL GO.\T BONE

~IARROX+

INTRODI*CTIOY

‘I%e ultrastructure of the eosinophil leucocytes and the development of their granules has been studied in the bone marro\v of several mammals and birds (Pease, 1956; Goodman, Reilly and Moore, 1957; Miller, Harven and Paladr. 1966; Wetzel, Horn and Spicer, 1967; Campbell, 1967; Hudson, 1970: Bainton and Farquhar, 1966; Scott and Horn, 1970; A\laxwell and Siller. 1972; Nirmalan, Atwal and Carlson, 1972). No such information on the eosinophil 1eucoc);tes of the goat (C’aprinae CU@Z vxists in the literature. The present stud!- \\.as, therefore, undertaken to investigate the granular development of the cosinophils of the bone marrow of’ the xoat.

MATERIALS

AND

hlETIIODb

Fcxmoral bone marrow, obtained immediately after death by electrocution from t clinically healthy laboratory goats of ages between 2 months and 24 years, was placed in already chilled fixative (2.5 per cent. glutaraldehyde in 0.2 M phosphatt. buff
RESULTS

The earliest recognized form of eosinophil granulocytic differentiation \vas rhe appearance of small immature granules which developed in close association \vitll the dilated rough endoplasmic reticulum I’RER). This form was in strict contrast to the granule development of the ncutrophil lcucocytes in which the‘ recognized manifestation of granule development takes place in the Golgi c,omples as described by Bainton and Farquhar 196ti ,.

184

0.

S. ATWAL

Early Stage The nuclei in the earliest recognized eosinophils wcrc large and indcntrtl with finely dispersed heterochromatin and identifiable nucleoli (Fig. 1). ‘I’hc~ membrane bound immature granules were spherical and contained an electroll dense homogenous material which, in some of the granules, did not complctel>r fill the whole rounded contour of. the outline membrane. The RER ~vas

Fig.

1. An early immature eosinophil containing a large nucleus (IX). The rough endoplastic reticuiun! is more dilated. The developing immature granules (IG) are intermingled with partially fillrd vacuoles (arrows) which contain less regular outlines. Prominent mitochondria (hi) and liw polyribosomes are quite apparent. PMN-neutrophil; Mac macrophage with phagosr,m~~\ and a Golgi complex (*). :.: 11 000.

ULTRASTRUCTURE

OF

THE

GOAT

EOSINOPHILS

185

prominent and showed a generalized dilation. In many instances the dilated portions of the RER contained a small amount of dense material. The fret polyribosomes were extensively distributed throughout the cytoplasm. A few autophagic vacuoles were present in the perinuclear zone. A modest number

l,‘ig.

2. Eosinophil of intermediate stage. The dilated cisternart of rough endoplastic reticulum are OI the branching type (arrows) which contain fine. moderately dense material. The mature, granules are lying around a very prominent Golgi’complex (GO). Small vesicles arising from the Golgi cisternae are seen fusing with the: granules. EOSimmature cosinophil with :f developing vacuole; PMN-neutrophil. i’ 10 000.

I:ig. 3. A mature eosinophil with a nucleus with clumping heterochromatin. fully differentiated crystalline core. A moderate amount ofrough less conspicuous Golgl complex iGO) in the vicinity of a centriolr :zwrow P) and a massive amount of ~ytoplxsm extending in the arc seen indicating nctivv motility oi nn eosinophil approaching mature granule (CR) sho\vs tubular componmts againrt :t dcnsr. basophil. ;.’ 10 000.

Alost granult-s contain ;I cndoplasmic reticulum and :I arc present. .\ pscudoporl forn~ of :I lobopotl :~~‘r~nv I. maturity. .I tra1nsvcrwl~ VLIL rimrrrpbour mntwi;ll. IS.\S

ULTRASTRCCTURE

I:iq.

OF

THE

GOAT

EOSINOPHILS

187

4. .\II

eosinophil with a bilobed nucleus, which c-ontains clumps of heterochromatin. AXumcro~~~ of round. oval, angular mature granules are evident. Thv crystalline cores are ot‘differrllr tixms. A few isolated rough endoplasmic reticulum tubules are scattered in the cytopla,nl ;\I -mitochondrion: lobopod-arrow L: EOS -portioll of another mature eosinophil whit t: contains gran~des with complex tubular and wsiculnr intvmum marrow (:I. .. 12 000. limns

of- small, rounded mitochondria were present. The developing immature granules intermingled with partially filled \racuoles \z,ith a less regular outline than the comparatively differentiated electron dcnsr spherical granules. The.

188 RER showed a focal dilation their development.

0.

s. AT\VAL

as the cells approached

the. intermcdiatc,

stagl;c 01

Intermediate Stage Developing eosinophils of the intermediate stage contained more dilattd profiles of RER than the earlier stage. The dilated cisternae were showing ;I branching pattern and contained finely distributed dense material (Fig. 2). ‘lIr% number of intermediate stages in a particular grid under examination wcrt few in comparison to either early or late mature stages. The granules were of rounded, oval, angular and irregular shapes in contrast to the earlier spherical forms. At this stage the characteristic crystalloid core of the granules appcarcct in various pleomorphic forms. The predominant variant was the tubular hlrtll which in cross-sections appeared like a collection of tubular mosaic arrays against the less dense, amorphous back-ground inside the outer mcmbranc of the granules. Prominent Golgi was the most characteristic feature of this sragc. (Fig. 2). Small, rounded vesicles emanating from the Golgi cisternac \V(‘IC sc(‘ti coalescing with the developing granules. Mature Stage The mature stage of eosinophil development \\-as recognized from ttlc nuclear lobation and the clumping of the heterochromatin into patches along the nuclear margins. No nucleoli were observed during the maturation stage. Modest amounts of RER were present in the form of orthodox isolated cisternac in contrast to highly dilated cisternal profiles observed in earlier stages. Thr Golgi complex was present although not very conspicuous. A few maturr forms showed centrioles in the perinuclear region. The extended portions of‘ cytoplasm in the form of pseudopods and lobopods were seen in the majorit). of the mature eosinophils (Fig. 3). The stretched portions of cytoplasm in these extensions was mostly organelle-free except for a few isolated RER tubules. .\ few long mitochondria were present in the area next to the portion of’ the cytoplasm occupied by the granules. All the granules had a characteristic central core which had already appeared in the intermediate stage (Fig. 41. DISCUSSION

The present study suggests that the granules of mature eosinophils of the goat bone marrow are derived from the small vacuoles tvhich contain ;I moderate amount of dense flocculent material. The configuration of thcsc vacuoles suggest a close association with RER, although the evidence of this association is not very convincing. A lack of a clear-cut indicatiotl of the association of these vacuoles with RER was also realized in previous studies (Wetzel et al., 1967; Scott and Horn, 1970; Hudson, 1970; Maxwell and Siller. 1972). However, it is clear that in the early development stages, there is a lack of vital association with the Golgi complex which plays a prominent role in the formation of granules of neutrophil leucocytes of mammals and birds (Bainton and Farquhar, 1966; Scott and Horn, 1970; Nirmalan et al., 1972). The appearance of a prominent Golgi complex during the intermediate

CLTRASTRUCTURE

OF

THE

GOAT

EOSISOPHILS

I 89

developmental stage and the arising array of small, dense vesicles c.oncurrenti> fusing with the granules, strongly suggest that the Golgi complex is indirectl) involved in the development of eosinophil granules, perhaps to incorporate materials into the granules in later stages of development M-hen characteristic. crystalloid cores become the obvious morphological fkms. Similar suggestions have been made by Wetzel et al., (1967) and Scott and Horn (1970) whcr~ describing the development of granules of rabbit and human eosinophil 1~Wcocvtes. The dense and highly organized crystalline cores of cosinophil granules ha5 I~en attributed to the presence of basic proteins and mucopolysaccharidcs (Horn and Spicer, 1964; Archer and Hirsch, 1963). Eosinophil granules contain the most detectable and abundant sulphated muco-substances iIIur111 and Spicer, 1969). It is conceivable that such materials are transported irlsidc the vesicles arising from the Golgi complex during the intermediate stage development bvhen crystalloid cores are beginning to appear. A process of’ accrc.tion and condensation of the contents of these vesicles results in organized, crystalline cores in the centre of the granules. The sulphate metabolism is thcs function of the Golgi complex. The enzymes required for the transfer of‘ sulpllates to a variety of acceptor molecules are located in the Golgi compler (Young, 1973). The prominence of the Golgi complex during the intermediatc~ stag< Lvhen crystalloid cores are appearing in the matured granules ma!signify the structural correlation of the transfer of inorganic ions and their transfer enzymes in the vesicles. Dru-ing the present study such tubular structures \\‘cre constantly obserl-cd in nlost of thr normal mature granules. It is concei\.able that the granules (11 the goat eosinophil ha1.e different enzymological properties or that the appearancc~ of tubular structures is a consequence of a fixation artefact, a phenomenon lb-hit h may be peculiar to the goat eosinophil. The preliminary cytochemical studies in this laboratory indicate that enzymological behaviour of goat cosillophil granules is perhaps fundamentally different from the other mammalian leucocJ.tes (unpublished data). The results of the present stud!. also suggest that steps involved in the differentiation of goat eosinophils and the, fbrnlation of their granules do not follow the classical pathkvays of the secretor) ;Ipp;u-atus in xvhich appropriate compartments (RER, Golgi complex and c,vrntually thr granules) are involved in a sequcntiul fashion in timr for tht synthesis and isolation of the proteins of thta grnnulcs ! ,Xcutra and I,cblontl. 196fi; Bainton and Farquhar, 1966).

The ultrastructure of the eosinophil leucocytes and the development of their granules were studied in the bone marrow of the goat (Cuprinae cupra). Thrcr distinct forms consisting of early, intermediate and mature stages \vcrc recognized. The granules developed from the dilated endoplasmic reticulum and assumed the mature crystalloid form in the early intermediate stage when the Golgi complex appeared very prominent. The mature granules were of sevc.ral shapes; the prominent variant contained tubular profiles against a less

190

0. s. .~\T\VAI~

amorphous background. The role of‘ a rough endoplasmic reticulum a~ld the Golgi complex in the development of the eosinophil granuloqtes is cornpared with their role in the classical pathways of the secretory apparatus. dense

ACKNO\VLEDGJIENTS

This investigation culture and Food. acknowledged.

was supported The technical

by funds assistance

from the Ontario Ministry of‘ Agriof Dr Thursa Wilson is gratefully

REFERENCES

Archer, G. T., and Hirsch, J. G. (1963). Isolation of granules from eosinophil leucocytes and study of their enzyme content. Journal of Experimental Medicine,

118,277-286. Bainton, D. F., and Farquhar, M. G. (1966). Origin of granules in polymorphonuclear leucocytes: Two types derived from opposite faces of the Golgi complex in developing granulocytes. Journal of Cell Biology, 28, 277-301. Campbell, F. (1967). Fine structure of the bone marrow of the chicken and pigeon. Journal of Morphology, 123, 405-440. Dunn, W. B., and Spicer, S. S. (1969). Histochemical demonstration of sulfated mucosubstances and cationic proteins in human granulocytes and platelets. Journal of Histochemistry and C’ytochemistr_y, 17, 668-674. Goodman, J. R., Reilly, E. B., and Moore, R. E. (1957). Electron microscopy of formed elements of normal human blood. Blood, 12, 428-442. Horn, R. G., and Spicer, S. S. (1964). Sulfated mucopolysaccharide and basic protein in certain granules of rabbit leukocytes. Laboratory Investigation, 13, 1 -15. Hudson, G. (1970). Ultrastructure of eosinophil leukocyte granules m the dog. .ilrfn anatomica, 77, 62-66. Maxwell, M. H., and Siller, W. G. (1972). Th e ultrastructural characteristics of eosinophil granules in six species of domestic bird. Uyournal of Anatomy, 112,

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E. D., and Palade. G. E. (1966). The structure of eosinophil E., Harven, leukocytes granules in rcdents and in man. Journal of Cell Biolog, 31, 349-362. C. P. (1966). Synthesis of the carbohydrate of mucous Neutra, M., and Leblond, in the Golgi complex as shown by electron microscope radioautography of goblet cells from rats injected with glucose-HH3. Journal of Cell Biolo~, 30, 119.-131. Nirmalan, G. P., Atwal, 0. S., and Carlson, H. C. (1972). UItrastructuraI studies on the leucocytes and thrombocytes in the circulating blood ofjapanese quail. Pot&y Science, 51, 2050-2055. Pease, D. C. (1956). An electron microscopic study of red boric marrow. Blood, 11, 501-526. Scott, R. E., and Horn, R. G. (1970). F’me structural features of cosinophil granulocyte development in human bone marrow: Evidence for granule secretion. Journal of Ultrastructure Research. 33, 16-23. Wetzel, B. K., Horn, R. G., and Spicer, S. S. (1967). Fine structural studies on the development of heterophil, eosinophil and basophil granulocytes in rabbits. Laboratory Investigation, 16, 349-382. Young, R. W. (1973). The role of the Golgi complex in sulfate metabolism. Journal of Cell Biology, 57, 175-189. Miller,

[Received.for publication,

3un.e 3rd, 19751