Ultrastructural changes of bone marrow megakaryocytes in several types of injury

Burns (1984)

10, 282-289

Printed in Great Britain

U ltrastructural changes of bone marrow megakaryocytes in several types .of injury Cheng Tian-Min,

Lin Yuan, Gu De-Quan, Luo Chang-Kun

and Zheng Huai-En

The Third Military Medical College, Chongqing, People’s Republic of China Summary

Seventy-one dogs were used in this study. Bone marrow tissues were examined by LM and EM and the peripheral blood platelets were counted. On the basis of previous research, megakaryocytophagia in bone marrow was confirmed. It was also proved that neutrophilic granulocytes participate in the autophagocytosis reaction in the body. Megakaryocytophagia plays a major role in the elimination of degenerate megakaryocytes in the marrow. The pathological change in megakaryocytes is considered to be one of the important causes of decrease in number and impairment of function of the platelets in bum, blast injury and burn-blast combined injury.

dogs were examined by LM ively. Smears from squeezed animals were also observed. platelet counts were performed

and EM respectmarrow of some Peripheral blood in 47 dogs.

RESULTS 1. Peripheral

count

blood platelet

There were no obvious changes in blood platelet counts in the blast injury group, but in the burn and burn-blast combined injury, the counts fluctuated to a lower level than the preinjured value for a long time (Fig. 1).

INTRODUCTION

In previous research, it was found that bone marrow megakaryocytes were killed and phagocytozed by granulocytes (designated as megakaryocytophagia) after burn, blast injury and bum-blast combined injury, as well as in the recovery stage of radiation sickness (Chang et al. 1963, 1980). The present study laid particular emphasis on the ultrastructural features and the significance of these changes. MATERIALS

AND

METHODS

A total of 7 1 dogs were used in this study: 24 suffered from bum-blast combined injury induced by nuclear explosion; 12 were exposed to flash radiation from a 5 000 W bromotungsten lamp to induce a 15 per cent TBS full thickness bum on the back; 18 were exposed to a blast wave created by a 200 g TNT charge explosion in a shock tube to induce blast injury; another 12, to both flash radiation and blast wave to induce bum-blast combined injury. In addition, 5 dogs served as normal controls. The bone marrow tissues (from sternum and femur) -of 7 1 and 44

. .. ... . aurn -Combined

I

_m P

I

5

10 15 20 Postinjured day

25

30

1. The changes of peripheral blood platelet counts in three different types of injury.

Fig.

2. LM examination of marrow There was no significant difference in the population of marrow nucleated cells among injured groups and between the injured and control animals. However, the proportion of megakaryocytes, as calculated by counting 5 000 nucleated cells in the marrow sections, was much higher than that of control group. The proportions of cells showing degeneration and phagocytosis by

283

Cheng et al.: Ultrastructuralchangesof bone marrow megakaryocytes Tab/a /. Proportion of megakaryocytes

in different groups Proportion

Groups

Nuclear explosion Burn-blast Laboratory Burn Blast Burn-blast Control

Cases observed

Megakaryocytes in nucleated cells

24

0.253

12 18 12 5

0.228+0.020* 0.196+0.009t 0.263 +O.O18t 0.136+0.008

Compared with control: *P
k 0.024*

Degenerative megakaryocytes

26.29 + 3.53t 55.3*7.5t 61.4+4.9t 63.17*3.46t 8.6kl.91

Phagocytized megakaryocytes

6.70+

1.56

14.25k3.4’ 7.0 12.34 13.75+3.11* I.6 iO.51

tP-cO.01

granulocytes in each 100 megakaryocytes were also increased significantly (Table I). The degenerative and phagocytozed megakaryocytes in sections and smears in different groups exhibited identical morphological features. The cells of the megakaryocytes belonged to the neutrophilic granulocytes, most of them were mature PMN, some band cells, metamyelocytes and even myelocytes, and occasionally eosinophils were found (Figs 2, 3). Near or surrounding the megakaryocytes, there were other haemapoietic cells in different stages of development (Fig. 4).

Fig.

3.

The cells inside a megakaryocyte, tens in

number, are essentially neutrophilic granulocytes. Smear from squeezed marrow, HE x 440.

3. Electron microscopy of marrow The ultras true tural changes megakaryocytes

Fig. 2. The degenerative megakaryocyte phagocytozed by granulocytes (black arrow) and the normal one is not (white arrow). HE x 440.

in degenerative

The ultrastructure of normal immature and mature megakaryocytes was as described by other authors. In the degenerative megakaryocytes, the nuclei were more lobulated and shrunken, with irregular, even saw-tooth like margins no nucleolus in general, clumps of condensed chromatin occurring near to the nuclear membrane (chromatin margination), sometimes

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Fig. 4. Around the megakaryocytes, there are various

kinds of haemapoietic cells. HE x 450. with iocal karyolysis. The outer nuclear membrane usually protruded outward, the perinuclear space became widened and confluent with the vacuolated SER. As for the cytoplasm, the demarcation membraneous system (DMS) and specific granules became disorganized in structure and diminished in number, scattered with vacuoles, myelin figures, autophagosomes, secondary lysosomes, residual bodies and swollen mitochondria with dispersed cristae, occasionally with finger print-like degeneration and whorl body formation of RER (Figs 5, 6). Some dilated vacuoles formed in the periphery of the cells in which no organelles or specific granules were present, but only amorphous fine granules and/or a few small vesicles could be found (Fig. 7). These degenerative changes may occur in different stages of development of megakaryocytes but more in mature cells. Both in nucleus

Fig. 6. A part of a megakaryocyte showing widening

of perinuclear spaces and disorganization of plasma components with formation of autophagosomes and vacuoles, as well as a decrease of specific granules. x12400.

Fig. 7. Vacuoles formed in the periphery of a mega-

karyocyte (arrow) in which no organelles or specific eranules were seen. onlv amomhous fine granules and small vesicles. x 5 000. a--------

-

and in cytoplasm, the changes occurred usually simultaneously or sometimes independently, scattered locally or all through the cell body. The ultrastructural megakaryocytes

Fig. 5. In a degenerated megakaryceyte, the nuclei

shrink with chromatin margin&ion and the perinuclear spaces become widened and confluent with dilated SER. x 8 000.

changes of phagocytozed

There were a series of features indicating the sequential processes of the entrance of granulocytes into the megakaryocytes. At first, the granulocytes, single or several in number, attached to the megakaryocytes by their pseudopodia or a lateral part of the cells (Fig. 8,9). The granulocyte pseudopodium protruded into the megakaryocyte, as evidenced by the morphological features and the absence of specific granules in the process (morphological characteristic of the forward motile pseudopodium

Cheng et al.: Ultrastructural changes of bone marrow megakaryocytes

Fig. 8. Several

plasmic

processes

of a neutrophil

(NEU) attaching to the membrane of a megakaryocyte (MEG). x 15 000.

Fig. 9. Lateral part of three neutrophils (NEU) attached closely to the megakaryocyte (MEG). x 8 400.

(Dorothea et al., 1964)). Then, a part of the granulocyte entered into the megakaryocyte, while, these two types of cells were still separated from each other by their own cell membrane (Figs 10, 11). Finally, the whole body of the granulocyte entered into the megakaryocyte, to a,varying depth. Within which most of the granulocytes showed an apparently normal appearance with intact plasma membrane and more granules (lysosomes), and no enveloping membrane could be seen around these cells (Fig. 12). At the surface of the granulocytes, there were one or more membrane invaginations and plasmic processes engulfing and ingesting the

285

Fig. 10. A granulocytic pseudopodium protrudes into the periphery of a megakaryocyte (arrow) in which no specific neutrophilic granules can be seen. Such a feature is characteristic for the advancing pseudopodium. x 10 000.

Fig. 11. A large part of two neutrophils invade into a megakaryocyte, but these 2 types of cells are still separated from each other by their own cell membrane. x 6 600.

megakaryocyte components (vacuoles, autophagosomes, secondary lysosome, etc.) and thereafter phagosomes were formed in the granulocytes (Figs 13-l 5). Granulocytic granules transferred nearby or attached to the cell membrane (in normal conditions, there always exist some distance between the granules and the cell membrane as reported by Dorothea et al. (1964) and examined in this study) and some granules penetrated through the membrane into the plasma of the megakaryocyte, where small

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Burns Vol. 1Of No. 4

Fig. 14. A neutrophil ingests the secondary lysosome of megakaryocyte (arrow). x 10 400. Fig. 12. The whole body of a neutrophil is situated inside a megakaryocyte. There is no membranous envelope around the neutrophil which is rich in granules and with many small membrane invaginations at the surface. x 10 920.

Fig. 15. Inside a degenerate megakaryocyte phago-

somes (arrow) are formed in a neutrophil, the structure of the phagocytozed vacuoles is just the same as that found in the plasma of a megakaryocyte. x 23 200. Fig. 13. Inside a megakaryocyte, a neutrophil ingests the former component (vacuole) by their plasma processes (arrow). x 10 600.

breakages of granulocytic membrane and lysis and disruption of megakaryocyte components could be found (Fig. 16). In others, this lysis or disruption also occurred surrounding or near the granulocytes, but no granules could be seen (Fig. 17). There might also be a number of granulocytes entering into the deeper part of the megakaryocytes, almost attaching to the nucleus (Fig. 18). Most of the phagocytozed megakaryocytes appeared to be degenerate, but others, possessed more or less structures of DMS and specific

granules which were responsible formation. DISCUSSION 1. Further confirmation phagia

for the platelet

of megakaryocyto-

The phagocytic action of the megakaryocytes and the platelets has been reported by some authors (Halstead et al., 1967; White, 1972), but in our studies, it appears that the megakaryocytes are phagocytozed by neutrophilic granulocytes after several types of injury. It is known that the megakaryocyte can protrude plasms to form a process in situ, but the cell body cannot move away (Kinosita et al., 196 1;

Cheng et al.: Ultrastructural

changes of bone marrow megakavocytes

Fig. 16. Inside a megakaryocyte, granules of a neutrophi1 are situated near by or attached to the cell membrane on which many small breakages exist, and some granules are discharged outside of the cell, where the megakaryocytic components are dissolved (arrows). x15000.

Fig. 17. Local lysis of megakaryocytic plasma appears close to a neutrophil (arrow) where no discharged neutrophilic granules can be found. x 13 750.

Lichtman et al., 1978). Either in sections or in smears of marrow, the cells inside the megakaryocytes were essentially neutrophilic granulocytes. As mentioned above, there were different series of haemopoietic cells surrounding the megakaryocytes in the marrow, thus, we have no reason to believe that the megakaryocytes, especially after degeneration, can move to phagocytoze the neutrophilic granulocytes selectively here and there. On the other hand, the granulocytes possess an active ability of migrating and ingesting.

287

Fig. 18. The entrance of three neutrophils into the deeper part (attach nearly to the nucleus) of a megakaryocyte x 8 000.

This fact suggests that the megakaryocytes are phagocytozed by granulocytes. Further direct evidence supporting the existence of megakaryocytophagia was provided by electron microscopy. The ultrastructure of the granulocyte pseudopodium within the megakaryocyte indicated that it was extending forward, the granulocytes entered into the megakaryocytes from the periphery to the depth, no membranous envelope was found around the inside of the granulocytes, most of the granulocytes remained intact and the megakaryocytes were degenerate or even dissolved, and the granulocytes were ingesting the megakaryocyte components and showing definite features of phagocytosis. Of course, there is no denying the phagocytic activity of megakaryocytes, but, according to the findings described above, bone marrow megakaryocytophagia undoubtedly exists after severe injuries. 2. The phagocytotic actions of the granulocytes inside of the megakaryocytes We found that inside the megakaryocytes, the granulocytes played a phagocytic function i;l three ways: (1) invagination of the cell membrane, ingestion of megakaryocyte components and formation of phagosomes; (2) discharge of lysosomal granules into the megakaryocyte, being considered as another type of degranulation; and (3) release of lysosomal content (hydrolases) into the megakaryocytes as proved indirectly by lysis of megakaryocyte components close to

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Burns Vol. l-O/No. 4

the granulocytes where no discharged granules could be seen. In addition, if the granulocytes broke down after death, the megakaryocyte would be dissolved simultaneously by the action of the lysosomal contents of the disintegrated granulocytes. 3. The significance

and influence

of mega-

karyocytophagia

(1) It is well known that autophagocytosis is an important biomedical phenomenon and the macrophages play a major role in this reaction. As for the neutrophilic granulocytes, designated as microphages, many studies focus attention on the phagocytosis of bacteria and small particles. In this paper, it is evident that the neutrophilic granulocytes also participate in the autophagocytosis in the body under certain situations. (2) Some papers have reported that the megakaryocytes were phagocytozed and cleared by macrophages in the marrow as they failed to form platelets (Thompson, 1977). In this study, no macrophages engulfing the megakaryocytes were seen by LM and EM, but megakaryocytophagia was an outstanding feature. The megakaryocyte lost its cell body gradually as the number of neutrophils increased inside the cell and finally, the structure of the megakaryocyte completely disappeared and there remained a stack of granulocytes. So, it is reasonable to suggest that the degenerate megakaryocytes were eliminated from the marrow mainly by way of megakaryocytophagia. The phagocytozed megakaryocytes would be carried out by the blood stream to the lungs and liver etc. and then eliminated (Fig. 19). (3) After severe bum or trauma, prolonged impairment of the function and a decrease in the number of blood platelets usually indicate a poor prognosis (Eurenius, 1972, 1979) but the mechanisms of these changes remains obscure. The following facts might be helpful for discussion of this problem. (1) The increased Table /I. The comparison the blood platelet count Blood platelets (% of preinjured value)

HE x 420.

proportions of degenerative and phagocytozed marrow megakaryocytes corresponded to the decrease of peripheral blood platelets in general, (Table ZZ). (2) The lifespan of canine platelets is about 7-9 days (Shi, 1980), so that the platelet counts on day 10 were analysed to indicate the platelets produced by marrow megakaryocytes after injury. In all eight fatal cases, the platelet count decreased to less than 50 per cent compared with the preinjured value, except in two dogs where the mean value was 44.5k5.65 per cent. Among the thirteen cases that were killed on postinjury day 30, the platelet counts in eleven dogs were more than 50 per cent (5 of them even more than 100 per cent). Their mean value was 86.38 & 12.9 per cent. It is thus evident that the platelet counts on the postinjury day 10 in fatally injured animals were significantly lower than in the animals that survived (P~0.05). (3) In animals with simple bums ofthe same degree, the proportions of both degenerative and phagocytozed megakarocytes were significantly different in animals that survived and those that died (Table ZZ). (4) Normally, the platelets are formed

between

the megakaryocyte

proportion and

Megakaryocyte proportion

Cases observed

Degenerative

Phagocytozed

9 18 15

68.89t-6.7 40.44A15.58 44.2 k7.27

14.78f3.57 10.28k2.09 7.47 f 1.92

<50+ 51 loot lOl-3824 Degenerative

Fig. 19. Megakaryocytes phagocytized by granulocytes are also found in the pulmonary vessels (arrow).

proportion between

??

and t P~0.01;

??

and 0 P~0.05

289

Cheng et al.: Ultrastructuralchangesof bone marrow megakaryocytes Tab/e III. Comparison of megakaryocytic changes animals with simple burris that survived or died

Group

Cases

Degenerative** proportion

Fatal

7

73.29k2.72

Survival

5

31.6

““P
by way cytoplasm peripheral

f9.63

in

the Burn Center in our college. The technical work was provided by the electron microscopy laboratory.

Phagocvtozed” proportion REFERENCES

21.14k4.24 7.0 k1.76

Cheng T. M. et al. (1963) Pathological changes in dogs that died in different stages after suffering from 400 R acute radiation sick&s. Symposium if Chinese National Conference Radiation Medicine,

“P
of separation of the megakaryocyte by DMS and breaking away of the plasma processes. The function of

platelets depended mainly on the organelles (e.g. specific granules) formed in the megakaryocyte (Constantinides, 1980). In this study, we found that the megakaryocyte degeneration and megakaryocytophagia may occur during different stages of development of these cells. In the degenerative and phagocytozed megakaryocytes, DMS were dispersed and specific granules decreased and even disappeared, and the peripheral processes or vacuoles were a result of a lack of organelles. Indeed, the function and the number of platelets not only depended on the condition of their parent cells (megakaryocytes), but also were influenced by many factors in the internal environment of the body. However, according to the facts discussed above, it is reasonable to conclude that the pathological changes in megakaryocytes which will influence their ability to form platelets is one of the important causes of a decrease in the number and impairment of function of the platelets in these injuries; and the degree of this change is one of the indications reflecting the outcome of disease, by becoming worse or improving. Acknowledgement

This paper was checked and amended by Dr. Li Ngao, Professor of Surgery and Chairman of Correspondence should be addressed to: Cheng Tim-Min, of China.

on Radiation

Biology

and

p. 38

Cheng T. M. et al. (1980) The bone marrow megakaryocytophagia in various injuries. Med. J. PLA. 5(6), 325, 328.

Constantinides P. (1980) Functional Electronic Histology, Translated by Liu Zhen Shan, Beijing, Scien&Publishing House, p. 6 1. Dorothea Z. P. et al. (1964) Electron microscooe studies on the degranulation of rabbit peritoneal leukocytes during phagocytosis. J. Exp. Med. 120, 569.

Eurenius K. (1972) Platelet and megakaryocytic kinetics following thermal injury, J. Lab. Clin. Med. 79,247.

Eurenius K. (1979) Hematologic changes in bums. In: Artz C. P. Moncrief J. A. and Pruitt B. A.(ed.) Burns A Team Approach Philadelphia, Saunders p. 132. Halstead L. S. et al. (1967) Phagocytosis by megakaryocytes. Blood 30, 543. Kinosita R. et al. (1961) Biodynamic of thrombonoiesis. In: Johnson S. A. (ed.) Blood Platelets London Churchill, p 6 11. . ’ Lichtman M. A. et al. (1978) The regulation of egress of cells from marrow In: Silber R., Lobue J. and Gordoni A. S. (ed.) The Year in Hematology New York Plenum. Shi X. Y. (1980) Animal Experiment Methods in Medicine. Beijing People’s Medical Publishing House, p. 395. Thompson R. B. (1977) Disorders of the blood, In: A Textbook of Clinical Hematoloav. YI Churchill Livingstone, p. “784. White J. (1972) Uptake of latex particles by blood platelets. Am. J. Pathol. 69, 439. Paper accepted 8 July 1983

The Third Military Medical College, Chongqing, People’s Republic