J Clin Pathol 1986;39:969-976

Pulmonary megakaryocytes: "missing link" betweencardiovascular and respiratory disease?G K SHARMA, I C TALBOT

From the Department ofPathology, University ofLeicester, Leicester Royal Infirmary

SUMMARY Pulmonary megakaryocytes were quantitated in a series of 30 consecutive hospital nec-

ropsies using a two stage immunoperoxidase stain for factor VIII related antigen. In all 30 cases theywere found with a mean density of 14 65 megakaryocytes/cm2 in lung sections of 5 gm in thickness.The maximum concentration of intrapulmonary megakaryocytes was consistently found to be in thecentral zone of the right upper lobe. Less than 22% of the observed cells possessed abundantcytoplasm, the rest appearing as effete, naked, and seminaked nuclei. The mean megakaryocytecount was found to be increased in association with both respiratory pathology (positive smokinghistory and impaired lung function) and cardiovascular disease states-shock; thromboembolism;myocardial infarction; and severe atheroma in the abdominal aorta, the coronary circulation, andthe circle of Willis. Pulmonary megakaryocytes probably embolise from bone marrow. This may

reflect stimulated thrombopoiesis, caused by increased platelet consumption in association withatherosclerotic disease, but it cannot be taken to confirm that the lung is the principal site of plateletproduction.

The megakaryocyte is now firmly established as thesource of all platelets, but the exact site and mech-anisms of platelet production and release remain indoubt.1 It is widely accepted that nascent platelets aredelineated within the megakaryocyte cytoplasm by ademarcation membrane system2 which forms byinvagination of the plasmalemma.3 This provides thelines of cleavage and will ultimately form the plateletplasma membrane. The actual release of platelets intothe circulation is explained by the "pseudopodialtheory"4'8 which proposes that a megakaryocyteproduces about six elongated cytoplasmic pseudo-ptdia, each with a core of platelet organelles. Thesepro-platelets penetrate the vascular sinusoids via thetransendothelial route and, after being pinched offinto the bloodstream, eventually fragment to yieldabout 1000 platelets. Recently, however, it was sug-gested that all platelet production occurs in the pul-monary microvasculature by the physical fragmen-tation of megakaryocyte emboli9 10 and considerableevidence supports this theory:1 The migration of intact megakaryocytes acrossthe marrow-blood barrier has been confirmed by bothstatic ultrastructural9 l and dynamic micro-cinematographic studies. 1 2

Accepted for publication 9 April 1986

2 Intact megakaryocytes are now known to benormal constituents of blood.133 Intrapulmonary megakaryocytes are a consistentfinding at necropsy,14 especially in disease states15and, despite early conflict of opinion," " vascularrearrangement experiments have proved that they donot arise de novo but are brought to the lungs by thevenous blood.184 The lungs serve as a filter for circulating mega-karyocytes because there is a reduction in both thenumber and size of megakaryocytes as blood flowsthrough the pulmonary microcirculation.9-245 Platelet production in the lungs is indicated byhigher platelet counts in central arterial blood than incentral venous blood. 16 22 23

The above evidence indicates that pulmonarymegakaryocytes are emboli from the marrow, which,due to their large size, are retained and fragmented inthe intrapulmonary capillaries to yield platelets.Agreement has not been reached, however, on theextent of their contribution to the total platelet popu-lation, with values from 7%25 to almost 100%21being reported. Recently, the platelet volume distri-bution, as measured by particle sizing, was shown tobe log normal rather than the Gaussian distributionusually shown by mitotic cells.26 It has been sug-gested that this unique volume distribution can be

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970explained only if all platelet production occurs in thelungs by a process of physical fragmentation, proba-bly as a result of sequential random binary divisionsat the pulmonary bifurcations.9 10 26 If this is the casethen disturbance of pulmonary-megakaryocyte inter-action may cause dysthrombopoiesis,27 which, inturn, may explain the common coexistence of cardio-vascular and respiratory disease.2830

This study aimed to explore these possibilities byinvestigating the incidence, concentration, distribu-tion, and morphology of pulmonary megakaryocytesin a series of hospital necropsies and relating thefindings to respiratory and atherothrombotic dis-orders.

Material and methods

A block of tissue 1-2 cm2 x 0-5 cm was collectedfrom the centre and periphery of each of the fourmain lung lobes during 30 consecutive unselected hos-pital post mortem examinations at the LeicesterRoyal Infirmary. Care was taken to avoid areas withlarge airways or obvious evidence of disease. After

Sharma, Talbot

overnight fixation in 5% formol/acetic acid (5%glacial acetic acid in 4% neutral phosphate bufferedformaldehyde solution) the tissue was paraffin pro-cessed and cut into 5 gm sections. These were thenstained for factor VIII related antigen, using anindirect immunoperoxidase technique, as describedby Crocker and Smith.3" Factor VIII related antigenis found on the platelet membrane,32 some of it beingadsorbed on to receptors from the plasma but a highproportion being an integral part of the plateletmembrane and originating from the demarcationmembrane system.33The area of each section was determined by plani-

metry using a Kontron Videoplan with graphic tabletbefore screening for megakaryocytes with a Leitz SM-Lux microscope, using the x 40 objective; all suspectcells were examined under oil immersion, using thex 100 objective. Megakaryocytes were categorisedinto three types: intact nuclei; seminaked nuclei; andnaked nuclei (fig 1). A fourth category, the "possi-ble", or "?" megakaryocyte, was also used to includecells satisfying some but not all the criteria neededfor the above three forms. Cells resembling

w.,

s~~~~~~. ..,

I

Fig 1 Morphology ofintrapulmonary megakaryocytes.

Fig la Intact megakaryocyte is large intravascular cell withlarge multilobed hyperchromatic nucleus enclosed byabundant positively staining cytoplasm.

Fig lb Seminaked megakaryocyte nucleus is similar butsmaller due to loss ofcytoplasm.

Fig lc Naked megakaryocyte nucleus is large intenselystaining nucleus, totally denuded ofcytoplasm, and mouldedinto shape ofenclosing vascular channel.

(DAB staining ofperoxidase-antiperoxidase preparations.)All magnifications x 320.

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971"Missing link" between cardiovascular and respiratory diseaseTable 1 Intrapulmonary distribution ofmegakaryocytes (mean valuesfor 30 subjects)

Site of tissue sampling No ofmegakaryocytes 2cm2_______________________ Absolute d!ffierence (%) difference

1 2 1 2 (1-2) (12 x 100) p value

Right lung Left lung 15-58 13-69 1-89 8-27 p < 0001Upper lobes Lower lobes 15-67 14-34 1-33 6-37 p < 0-005Central zones Peripheral zones 16-41 14-57 1-84 6-66 p < 0-0005

megakaryocytes were occasionally seen in the extra- CARDIOVASCULAR SYSTEMvascular spaces. These could be either true mega- Shock Shock was diagnosed from a clinical descrip-karyocytes or large alveolar macrophages and so were tion of pallor, sweating, tachycardia, and hypo-included in the "?" category. After attending the tension (100/70 mm Hg being taken as an arbitrarynecropsy and studying the clinical case notes three lower limit for the normal blood pressure) at somesets of clinicopathological data were recorded. stage during the week before death.

Thromboembolism The presence or absence ofRESPIRATORY SYSTEM thromboembolic disease was determined solely fromThe subjects were divided into smokers and non- the necropsy findings.smokers, and pulmonary performance was classified Myocardial infarction Infarcts found at necropsyas normal or impaired according to the clinical his- were described as being small (less than 1 cm2),tory, physical signs, and lung function tests (if per- medium (1-3 cm2), or large (greater than 3 cm2 orformed). transmural).

Table 2 Pulmonary megakaryocyte counts related to various clinicopathological data

Megakaryocytesfcm2 in sections oflung

StandardClinicopathological information No ofsubjects Mean Range deviation p value

Smoking Non-smoker 14 12-96 3-59-36 71 7-39 p = 0-03Respiratory history Smoker 13 18-04 1-71-29-66 7-15

system Lung Normal 7 13-53 1-71-21-00 6-23 p > 0-1function Impaired 22 15-57 3-59-36-71 7-90

Shock Absent 23 12-97 1-71-29-66 7-17 p = 0-02Present 7 20-12 12-15-36-71 7-40

Cardiovascular Thromboembolic Absent 16 13-22 1-71-29-66 7-65 p > 0-1system disease Present 14 16-25 3-78-36-71 7-72

Myocardial Absent 23 13-24 1-71-29-66 7-00infarction Small 1 36-71

Medium 1 18-20 p = 0-025Large 3 16-35 12-15-22-19 4-25

Sex Male 21 14-96 4-01-29-66 6-53 p > 0-1Female 9 12-33 1-71-36-71 10-90

Infection Absent 15 15 00 1-71-36-71 8-85Present 12 13-93 4-01-29-66 7-25 p > 0-1Blood-borne 3 15-60 13-17-19-53 2-81 p > 0-1

Malignancy Absent 16 15-80 1-71-36-71 8-96Present 6 12-29 3-78-23-32 7-64 p > 0-1Metastatic 8 14-07 6-95-19-53 4-30 p < 0-1

Miscellaneous Haemoglobin Normaldata M = 16 + 2g/dl 12 17-01 3-59-36-71 9-42

F = 14 ± 2g/dlLow 16 14-35 4-01-22-46 5-83 p > 0-1

White Normal 7 20-38 14-72-36-71 7-53cell 7 + 3 x 109/1count High 20 14-01 3-95-29-66 7-11 p < 0-05

Platelet Normal 17 16-98 3-59-36-71 8-19count 290 ± 150 x 109/1

Low 4 11-98 6-95-22-19 6-99 p > 0-1High 7 13-85 4-01-22-46 5-91 p > 0-1

Fever Absent 12 16-84 3-59-36-71 8-62Present 17 14-27 4-01-29-66 7-02 p > 0-1

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0 1 1-2 2-3 3-4 4-SGrade of aortic atheroma

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Mean 2-86 18-86 814(SD) 1.15 10-80 1-88

Fig 2 Pulmonary megakaryocyte counts and aortic athero

Aortic atheroma The abdominal aorta was openedlongitudinally and atheromatous severity was

assessed, as described by McGill et al,34 by com-

paring it with a panel of aortas graded 1-2 to 6-7inclusive. Four of the 30 cases studied could not besatisfactorily equated with any of the six specimenaortas; two were designated as grade 1 atheromawhile the other pair (both from newborn babies) were

classified as grade 0 atheroma.Coronary atheroma The main coronary arterieswere cut transversely at regular intervals and theluminal diameter assessed. Four grades of athero-matous severity were recognised: absent or all maincoronary vessels widely patent; mild, or minimalluminal narrowing of one of the major arteries; mod-erate or less than 50% luminal reduction of one or

more of the major coronary arteries; severe, or greaterthan 50% reduction of the luminal diameter of allthree main coronary arteries.Cerebral atheroma Atherosclerotic disease in thecircle of Willis was absent (widely patent), mild(noticeable luminal reduction), or severe (lumen morethan 50% occluded).

MISCELLANEOUS VALUESThe following miscellaneous clinicopathological datawere also recorded: sex, age, infection (absent,present, or blood borne), malignancy (absent,present, or metastatic), and the antemortem haemo-globin, white cell count, platelet count, and bodytemperature.

15-66 18-01 17-61 21-885-13 0 11-99 0-63

Results

THE INCIDENCE AND CONCENTRATION OFPULMONARY MEGAKARYOCYTESMegakaryocytes were found in the lungs of all 30 nec-ropsy specimens studied, but their concentration wasvariable, ranging from only 2 megakaryocytes/cm2to as many as 37 megakaryocytes/cm2 (mean 14-65megakaryocytes/cm2).

40-

30-

MKs/crn2 20-

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I p=0o05r- p = 01- ,r-p >0.1 -_

Absent Mild ModerateSeverity of coronary atheroma

Severe

Mean 10 61 11-06 11-98 18-51(SD) 8-66 551 5-11 8-19

Fig 3 Pulmonary megakaryocyte counts and coronaryatheroma.

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30-

MKs/cm2 20-

10-

I p>OI

I . I

I

iI

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Absent Mild Severe

Severity of cerebral atheromaMeon 11*30 15-87 17-23(SD) 7-74 7 82 4 10

Fig 4 Pulmonary megakaryocyte counts and cerebralatheroma.

THE INTRAPULMONARY DISTRIBUTION OFMEGAKARYOCYTESTable 1 shows that there is a higher concentration ofmegakaryocytes in the right lung than in the left, inthe upper lobes than in the lower lobes, and in thecentral zones than in the periphery.

THE MORPHOLOGY OF PULMONARY

MEGAKARYOCYTESIn all parts of the lungs only 22% of megakaryocyteswere intact, with copious cytoplasm. Thirty one percent were seminaked nuclei, 31% were naked nuclei,and the remaining 16% were classed "?".

PULMONARY MEGAKARYOCYTES AND

CLINICOPATHOLOGICAL RELATIONS

Respiratory system Pulmonary megakaryocyteswere slightly increased in association with both a

positive smoking history and impaired respiratoryfunction, though significance was reached only in thecase of a positive smoking history (table 2).Cardiovascular system There was a significantincrease in the pulmonary megakaryocyte count inassociation with shock, myocardial infarction, andsevere atheroma in the abdominal aorta, coronarycirculation, and the cerebral vessels. The increase inpulmonary megakaryocytes in thromboembolic dis-ease was only slight (table 2 and figs 2, 3, and 4).Miscellaneous clinicopathological data The pul-monary megakaryocyte count was significantlyhigher in relation to leucocytosis and slightly higherin men than in women, but there was no obvious asso-

ciation with age, infection, neoplasia, haemoglobinconcentration, platelet count, or fever (table 2).

Discussion

The incidence of pulmonary megakaryocytes was100%, thereby supporting the results of previouswork.'4 This seems to be correlated with the generalcondition of patients before they die. One otherstudy reported pulmonary megakaryocytes in 95% ofhospital necropsies but only 67% of forensicnecropsies."The concentration of pulmonary megakaryocytes

(14.65 megakaryocytes/cm2 in 5 pm sections) coin-cides closely with the results of Smith and Butcher,"5who counted 100 high power fields and reportedmean values of 14 2 megakaryocytes/mm3 for theirhospital cases but only 5-1 megakaryocytes/mm3 fortheir forensic series. Aabo et al,35 however, quote anaverage of 4 megakaryocytes/cm2 in forensicnecropsies and 37 megakaryocytes/cm2 in hospitalnecropsies in 7 pm lung sections and estimate themaximum density in normal lungs to be 18megakaryocytes/cm2.More recently, a study of burns victims found less

than 2 megakaryocytes/cm2 in lung sections ofunspecified thickness.36 There are two possible rea-sons for this variation in the reported concentrationof pulmonary megakaryocytes. Firstly, the pul-monary megakaryocyte count may be greater inhospital deaths than in forensic cases' 35 due to aneffect of disease states such as acute infections, 15 35 37leucocytosis,35 haemorrhage, 35 postoperativestates,37 38 shock,35 and the adult respiratory distresssyndrome.39 Consumptive coagulopathy is believedto be the common pathogenetic mechanism produc-ing increased pulmonary megakaryocyte counts inthese states.3540 Secondly, previous studies haverelied entirely on morphology for the identification ofmegakaryocytes in sections stained with haema-toxylin and eosin or periodic acid Schiff. Wells et a136explained their low pulmonary megakaryocyte countsby their use of more stringent criteria to define amegakaryocyte. In this respect our investigation morereliably indicates the intrapulmonary megakaryocytedensity, because it not only uses an immu-nocytochemical stain to assist in identification, but italso clearly defines the morphological criteria neededto identify a megakaryocyte.

The distribution of megakaryocytes is not uniform,and there is an appreciably greater concentration ofcells in the right lung than in the left. This contrastswith the results of previous work,4' and there is noobvious explanation. It could be a simple "poolingeffect"; perhaps right handed subjects are more likelyto lie on their right side. The right lung may receive adisproportionately greater share of the cardiac out-put, or, because of the phenomenon of "plasma skim-ming", the left pulmonary artery, which arises as an

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974offshoot from the main trunk, may receive only diluteplasma. Unknown local factors may make mega-karyocyte filtration more effective or fragmentationless efficient in the right lung, or both.The upper lobes have a consistently greater mega-

karyocyte count than the lower lobes, thus supportingthe conclusions of Bendix-Hansen et al,4' who expla-ined this in terms of bed rest changing the pulmonaryblood flow. Our observations also showed that pneu-monic consolidation was mostly confined to the lowerlobes while emphysematous changes were almostexclusive to the upper lobes.Emphysema may either cause an actual increase in

intrapulmonary megakaryocytes or simply facilitatetheir identification; the converse is true for pneu-monia. These two factors may be interrelated assevere basal congestion tends to divert blood to theupper lobes.

Consistent with the findings of previous work,41the central zones have a greater megakaryocyte countthan the lung periphery. Again, differential bloodflow is a likely explanation, although the greaterdegree of expansion and contraction undergone byperipheral lung tissue may also be important factors.

PULMONARY MEGAKARYOCYTES ANDRESPIRATORY AND CARDIOVASCULARPATHOLOGYThe common coexistence of pulmonary and athero-thombotic disorders is well known42 43 and cigarettesmoking has been implicated in both chronic bronchi-tis and coronary artery disease. The causal factors inthese disorders often coexist-cigarette smokers aremore likely to consume a high fat diet-these beingestablished risk factors in respiratory and cardio-vascular pathology, respectively. When Friedman etat44 failed to account for their observed associationbetween diminished vital capacity and clinical coro-nary artery disease in terms of these accepted expla-nations, they suggested the existence of "some otherconnection between pulmonary function and the stateof the coronary arteries or myocardium."

There are three such possibilities: firstly, chronicrespiratory disease may cause hypoxia whichincreases aortic cholesterol accumulation, probablydue to ischaemic endothelial injury45; secondly, lungdisease may impair intrapulmonary production ofprostacyclin production, thereby disturbing the nor-mal arterial homeostasis between prostacyclin andthromboxane A2 and thus predisposing toatherothrombosis4b; thirdly, if pulmonary mega-karyocytes are the principal source of platelets thendisturbance of the lung and megakaryocyte inter-action (due to a change in either the pulmonary ves-sels and their biochemical environment or in themegakaryocyte size and protein structure) may result

Sharma, Talbotin dysthrombopoiesis27 and thus predispose to bothrespiratory and cardiovascular disease.28 - 30 Ourresults indicate an increased pulmonary mega-karyocyte count in association with both respiratorydisease and cardiovascular disease, especially shockand thromboembolism (both findings agree withthose of previous reports15 35 37), myocardialinfarction, and severe atheroma.

There are two explanations for these observations.Firstly, the number of pulmonary megakaryocytesmay be increased in association with respiratory dis-ease, with resulting secondary atherothrombotic com-plications. It has already been shown that theefficiency of the pulmonary vascular filter can beaffected by lung diseases such as bronchitis and bron-chopneumonia.47 Impaired megakaryocyte fragmen-tation in certain respiratory disorders may yieldlarger fragments than usual-some of these may beretained in the lungs and appear as naked or semi-naked nuclei, while others will escape into the periph-eral circulation. As large platelets may be more reac-tive,48 50 this would predispose to secondaryatherothrombosis. Supporting evidence comes fromreports of an increased mean platelet volume inrelation to both chronic hypoxia30 and myocardialinfarction. 5l - 54 Interestingly, only 22% of the mega-karyocytes that we observed in the lungs possessedcopious cytoplasm, thereby suggesting that most areeither released from the marrow as effete naked andseminaked nuclei, or that intact cells emobolising tothe lungs are rapidly fragmented in the pulmonarycapillaries. This explanation seems unlikely, however,as the relative proportion of naked and seminakedmegakaryocyte nuclei in patients with severe ath-eroma was not increased. A more likely association isthat the number of pulmonary megakaryocytes isincreased in association with atherosclerotic diseasebecause of enhanced thrombopoiesis due to increasedplatelet consumption-either as a result of a gen-etically or environmentally acquired tendency, orboth, to increased platelet-vessel wall interaction, oras a result of increased platelet adherence to ulceratedatheroma. Respiratory complications may sub-sequently develop as a direct consequence of theincreased presence and fragmentation of mega-karyocytes in the lungs, a feature already implicatedin asthma,55 56 pulmonary hypertension, 57 the shocklung36 and the adult respiratory distress syndrome.39The higher pulmonary megakaryocyte counts

found in men are consistent with a higher incidence ofpulmonary and cardiovascular disease also found inmen. The failure to show a correlation between themegakaryocyte counts before and after death,although consistent with that of previous reports,35 36is not consistent with the hypothesis that the lungs arethe principal site of thrombopoiesis. The pulmonary

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"Missing link" between cardiovascular and respiratory disease

megakaryocyte content may be disturbed by the cir-culatory changes associated with death, a view sup-

ported by the fact that there is a direct relationbetween the circulating platelet count in life and thepulmonary megakaryocyte density; the introductionof thrombocytopenia in experimental animalsincreases both circulating and intrapulmonary mega-

karyocytes.4058 Previous reports indicate that thepulmonary megakaryocyte density is affected by bothhaemorrhage" and hypoxia.30 Our findings inrelation to shock agree with this. There was no

relation, however, between the megakaryocyte num-

bers in the lung and the haemoglobin concentration.In contrast to the findings of previous work,35 we

found no appreciable increase in the pulmonarymegakaryocyte count in association with fever, infec-tion, or neoplasia, though leucocytosis did have a

positive effect. Similarly, despite increasing athero-matous disease with advancing age there was no

demonstrable increase in the pulmonary mega-

karyocyte count. This, however, probably reflects age

clustering, as most of the subjects were of late middleage (mean age = 59 2 years, (SD 22 33)). Our findingssuggest that, although the lungs may not be the majorsite of platelet production, it would be wise not toneglect the role of pulmonary megakaryocytes inthrombopoiesis, particularly in relation to cardio-vascular and pulmonary diseases.

This work formed part of an intercalated BSc project,during which GS was supported by an MRC Award.We thank Mrs J Jones for secretarial help.

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Requests for reprints to: Dr IC Talbot, Department ofPathology, Clinical Sciences Building, Leicester RoyalInfirmary, PO Box 65, Leicester LE2 7LX, England.

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