The Journal of Clinical InvestigationVol. 46, No. 12, 1967

Effect of Chloroquine on Morphology of CytoplasmicGranules in Maturing Human Leukocytes-an

Ultrastructural Study *MARTHAFEDORKO

(From The Rockefeller University, New York)

Abstract. Bone marrow and peripheral blood from patients who had receivedchloroquine phosphate were studied to determine the effect of this drug on theultrastructure of cytoplasmic granules in leukocytes. Neutrophils from ap-proximately one-half of the patients who were treated developed abnormalcytoplasmic granules. Vacuolar, lamellar, and particulate components withinabnormal, large granules were present in myelocytes from certain patientswho received chloroquine therapy. Mature neutrophils and lymphocytesfrom these patients showed variable numbers of large, membrane-boundedstructures containing myelin figures. Cytoplasmic granules in eosinophilicmyelocytes from patients treated with chloroquine did not contain the usualcrystalloid structure, but instead contained small whorls of osmiophilic ma-terial. The granules in abnormal mature eosinophils were replaced by largevacuoles which contained amorphous material. The abnormal granules seenin these various white cells after chloroquine therapy may either reflectdefective granule formation or autophagy.

Introduction

Previous work by others has helped to establishthe true nature of cytoplasmic granules normallyfound within leukocytes. Extensive biochemicaland functional studies have demonstrated that suchcytoplasmic granules are lysosomes (1, 2).

Although stained smears of human leukocyteshave been studied for approximately 100 years,very little is known about abnormal leukocytegranulation. Abnormal granules in human leuko-cytes have been observed in both hereditary andacquired disorders. In the case of the latter, theetiology is diverse and the leukocyte abnormalityis often reversible. In preparations stained by theWright-Giemsa method normal human neutrophilgranules are pink-to-purple structures approx-

* Received for publication 5 June 1967 and in revisedform 24 July 1967.

This work was supported by grants AI 01831 and FR00102 from the U. S. Public Health Service, NationalInstitutes of Health, Bethesda, Md.

Address requests for reprints to Dr. M. Fedorko, TheRockefeller University, 66 Street and York Avenue, NewYork, N. Y. 10021.

imately 0.3 ,u in diameter, whereas the so-called"toxic" granules appear as 0.5-1 /x basophilicgranules. The ultrastructure of any type of ab-normal acquired leukocyte granulation has notbeen described and the pathophysiology of thechanges is unknown.

During the course of a study on human marrow,abnormal, large basophilic granules in neutrophilicleukocytes were noted on a smear from a patientwho received a course of chloroquine phosphate,7-chloro-4- ( 4-diethylamino - 1 -methylbutylamino)quinoline phosphate, therapy for sarcoidosis."Toxic" granules have been noted previously onblood smears from patients who have been treatedwith chloroquine (3). This communication willdescribe the ultrastructural changes in marrow andperipheral blood leukocytes from patients whoreceived chloroquine phosphate therapy.

Methods and MaterialsAdministration of drug. Six patients with sarcoidosis

were given 250-500 mg chloroquine phosphate (Win-throp Laboratories, N. Y.) orally, daily for periods of2-6 months. Diagnosis was confirmed in all patients in

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EFFECT OF CHLOROQUINEON HUMANLEUKOCYTES

the study by positive Kveim tests and clinical history com-patible with sarcoidosis.

Six patients with sarcoidosis who received no therapywere also studied.

Preparation of tissue specimens. Serial samples of1)eril)heral blood were collected from all patients before,(luring, and after treatment with chloroquine and wereprepared for electron microscopy in the following manner.Blood was collected into heparinized capillary tubes(Yankee, Clay-Adams, N. Y.) and centrifuged at 2500 gfor 15 min. The glass capillary tube was broken abovethe buffy coat layer; the buffy coat was then expressedinto fixative.

Bone marrow was aspirated from the posterior iliaccrest of one patient during and 2 months after a courseof chloroquine therapy. The marrow was collected intoa syringe coated with normal saline which containedheparin (Panheparin, Abbot, Chicago, Ill.) in a finalconcentration of 1.5 mg/ml. The marrow suspension wasthen centrifuged at 250 g for 5 min. Small particlesof marrow which contained fat rose to the top of theplasma, were aspirated, and then were either preparedfor light microscopy or fixed for electron microscopy.

Light nmicroscopy. Smears of both peripheral bloodand bone marrow particles were air-dried and stainedwith Wright-Giemsa stain.

Fixation for electron microscopy. Specimens wereeither fixed 1 hr at room temperature or overnight at40C in 2.5% glutaraldehyde in 0.1 NI phosphate buffer pH7.4, made by diluting 1: 20 a stock solution of 50% glu-taraldehyde (Fischer Scientific Company, Pittsburgh,Pa.) The specimens were then washed twice in coldisotonic saline for 30 min and postfixed in 1% osmiumtetroxide in 0.1 3ir phosphate buffer for 1 hr in the cold.Blocks were washed twice in cold isotonic saline beforebeing dehydrated in graded alcohols and embedded inEpon (4). The micrographs which appear in this paperare from cells which have been fixed by the abovemethod. Some specimens were also fixed in 1% osmiumtetroxide in 0.1 mr phosphate buffer pH 7.4 for 1 hr at 40Cand then received three 15-min washes in cold isotonicsaline before dehydration and embedding.

Electron microscopic localization of acid phosphatase.10 ml of heparinized blood was sedimented for 1 hr. Thewhite cell-rich plasma was separated off and centrifugedat 400 g for 5 min. The cell pellet was fixed by suspend-ing the cells for 15 min in 1.251% glutaraldehyde in caco-dylate buffer, washed twice in 0.25 AI sucrose, and thenincubated for 1 hr at 370C in 5 ml of prewarmed Gomorisubstrate pH 5.0, which had been freshly prepared with,3-glycerophosphate (Eastman Kodak, Rochester, N. Y.)The cells were then washed once and postfixed for 30 minin 1%c osmium in cacodylate buffer. After osmium fixa-tion the cell pellet was washed twrice for 30 min in coldsaline before dehydration and embedding. Cells incu-bated with inhibitors or without substrate failed to showreactivity.

All blocks were sectioned on a Porter Blum microtomewith a diamond knife, stained with uranyl acetate andlead citrate (5), and examined in a Siemens Elmiskop I.

Results

Abnormal granules in peripheral blood leuko-cytes occurred in three out of seven patients withsarcoidosis who received 250-500 mg of oralchloroquine phosphate daily for 2-6 months.Seven patients with sarcoidosis who received notherapy did not exhibit any abnormality in leuko-cytes. In one patient the ultrastructural changeswere present in peripheral blood and bone marrowleukocytes during two successive 3-month coursesof chloroquine therapy. After cessation of chloro-quine therapy the numbers of abnormal cells inperipheral blood progressively diminished. 1 monthafter the drug was discontinued, the ultrastructureof all leukocytes in blood and bone marrow wasnormal. During the course of therapy the whiteblood cell count in the peripheral blood of one pa-tient decreased from 5200 to 2800 and becamenormal after cessation of therapy. The other twopatients had normal blood counts during therapy.The patients who were receiving cholorquinephosphate and who demonstrated cytologic changesin leukocytes were not observed to have increasednumbers of infections, gastrointestinal distress, ordifficulties with vision.

Neutrophils. Mature neutrophils from bloodor bone marrow of sarcoidosis patients who hadreceived no therapy were identical in ultrastructureto cells obtained from normal persons (Fig. 1).The cytoplasmic granules appeared as round, ob-long, or biconcave electron-opaque structureswhich varied in size and shape. They were mem-brane bounded and for the most part did notpossess any internal structure, but occasionallycontained a crystalloid inclusion. Variable amountsof glycogen, sparse amounts of rough endoplasmicreticulum, and occasional mitochondria were pres-ent in the cytoplasm. On a stained smear themature cells showed typical small 0.2-0.5 u pink-to-purple granules (see insert, Fig. 1). The ultra-structure of an abnormal mature neutrophil fromperipheral blood of a patient treated with chloro-quine is seen in Fig. 2. Many of the cytoplasmicgranules were normal in appearance hut therewas striking alteration in the ultrastructure ofother cytoplasmic granules. The abnormal gran-ules were larger than the normal ones, and theypresented an internal structure of layered mem-branous elements (myelin figures). These myelin

1933

MARTHAFEDORKO

figures were also observed after the fixation withosmium alone. Identical changes were seen inmature neutrophils from bone marrow. Maturecirculating neutrophils contained occasional, largeabnormal granules which were positive for acidphosphatase after enzyme histochemistry was per-

formed at the ultrastructural level. The increasedsize and intense staining of the cytoplasmic gran-ules on a smear is demonstrated in the insert inFig. 2.

Bone marrow myelocytes from sarcoidosis pa-tients who did not receive chloroquine and from

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FIG. 1. MATURENEUTROPHILFROMA PATIENT WITH SARCOIDOSIS WHORECEIVED NO THERAPY.Shown are two lobes of the nucleus and abundant cytoplasmic granules which vary in size andshape. The appearance is identical with that of neutrophils from normal persons. X 21,000.The inset depicts the light microscopic appearance of a normal human neutrophil which wasstained by the Wright-Giemsa method. The lobulated nucleus and pale-staining granules areseen. X 1900.

1934

EFFECT OF CHLOROQUINEON HUMANLEUKOCYTES

4

FIG. 2. MATURENEUTROPHIL FROM A PATIENT WITH SARCOIDOSIS WHOWAS RECEIVING

CHLOROQUINEPHOSPHATETHERAPY. The cytoplasm shows many large granules containingmyelin figures (arrows). Apparently normal granules are also present. X 22,000. Theinset depicts the light microscopic appearance of a neutrophil from a patient during treat-ment with chloroquine phosphate. Lobes of the nucleus are visible as well as the large, in-tensely staining cytoplasmic granules. Method of preparation and stain is identical with thatin Fig. 2. X 1900.

normal persons contained membrane-bounded cyto-plasmic granules which did not have any internalstructure (Fig. 3). Smooth and rough endo-plasmic reticulum, often showing dilated cisternae,was widely distributed in the cytoplasm. TheGolgi complex occupied a relatively small area in

human myelocytes; vesicular, vacuolar, and lamel-lar elements were seen. A myelocyte from a pa-tient who was receiving chloroquine therapy isshown in Fig. 4. The affected granules wereabnormally large and contained internal structurewhich consisted of vacuolar, lamellar, and par-

1935

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MARTHAFEDORKO

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FIG. 3. NORMALHUMANMYELOCYTE. There are cytoplasmic granules which are membranebounded and without inclusions. Scattered elements of rough and smooth endoplasmic reticu-lum (RER) are present in the cytoplasm. X 33,000.

ticulate inclusions. In some instances these com-ponents appeared fused to the periphery of theabnormal granule. Another abnormal myelocyteis shown in Fig. 5. Several granules had incor-porated vacuolar elements and there was someevidence of early myelin figure formation.

Eosinophils. Normal mature eosinophils fromuntreated patients with sarcoidosis or from normal

persons contained cytoplasmic granules with thecharacteristic internal crystalloid (6). Normaleosinophilic myelocytes contained two types ofgranules: those without any internal structure andthose with a crystalloid inclusion.

The ultrastructure of mature, circulating eosino-phils from certain sarcoidosis patients treated withchloroquine was profoundly affected. The abnor-

1936

EFFECT OF CHLOROQUINEON HUMANLEUKOCYTES 1937

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FIG. 5. BONEMARROWMYELOCYTEFROMA PATIENT WHOWASRECEIVING CHLOROQUINE. The Golgi com-

plex (arrows) and scattered elements of rough endoplasmic reticulum (RER) are present. Granules(Ge) and (Gs) appear to contain vacuolar components. Granule Gs contains particulate elements, whereasG4 appears to be composed of lamellae. X 35,000.

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FIG. 6. EoSINOPHILIC MYELOCYTEFROMBONEMARROWOF A PATIENT WHOWASRECEIVINGCHLOROQUINETHERAPY. Normal cytoplasmic granules show either a crystalloid inclusion(not illustrated) or a homogenous dense matrix (G.), whereas the abnormal granules con-tain dense osmiophilic whorls (GC,-). X 27,000.

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PHATE THERAPY. A large membrane-bounded inclusion (of lamellar and particulate components is present within t

bounded myelin figures of various sizes. Someof the abnormal cytoplasmic structures were quitelarge (Fig. 7). These structures were positive foracid phosphatase. Except for these cytoplasmic in-clusions, ultrastructural characteristics were thosecommonly found in lymphocytes. Lymphocytes insmears of blood and bone marrow from these pa-tients contained occasional azurophilic granules.Evaluation of structural changes in histiocytes andmonocytes was not included in this study becauseof the difficulty in identifying their precursors inbone marrow specimens on the basis of morphologyalone.

DiscussionThe results presented here demonstrate that

chloroquine phosphate can exert a profound effecton cytoplasmic granules in immature and maturehuman leukocytes. The ultrastructure of acquiredabnormal leukocyte granulation produced by thisdrug has been described. Previous work by otherswould seem to indicate that chloroquine is incor-porated into leukocytes (3). The ultrastructural

'EIVING CHLOROQUINEPHOS-

(arrow) which is composedthe cytoplasm. X 23,200.

changes are probably, therefore, a direct conse-quence of a form of chloroquine which is incor-porated into cells.

The gradual diminution in the numbers ofabnormal cells after cessation of chloroquine ther-apy indicates that the drug effect is reversible butgives no information on the mechanisms involved.Evaluation of the reversibility of the chloroquineeffect on myelocytes under in vitro conditions isnot feasible, for there is ultrastructural evidenceof cell degeneration after 6 hr exposure to tissueculture conditions.1 The lifespan of circulatingneutrophils and eosinophils has been shown to berelatively short (4 days) (7, 8). On the basis ofthe data reported here, the reversibility of thechloroquine effect on granulocytes within theweeks following withdrawal of the drug is likelydue to replacement of the abnormal cells by anormal cell population. The presence of some ab-normal granulocytes for weeks after drug with-drawval is probably related to the fact that chloro-

1 Dr. M. Fedorko, Personal observations.

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EFFECT OF CHLOROQUINkON HUMANLEUKOCY8ES

quine is stored in body depots for variable periodsof time (9). On the other hand, lymphocytes,longer-lived cells (10), may either be able toreverse the cytologic effects of chloroquine or havea shortened lifespan. In the latter case there couldperhaps be replacement by morphologically nor-

mal lymphocytes.The hydrolases within lysosomes can digest

either exogeneous particles taken up by the cellor endogeneous material within the cell. Previousstudies have demonstrated the function of hydro-lytic enzymes within specific cytoplasmic granulesof neutrophils in attacking bacteria and other par-

ticles ingested by phagocytosis (2). Others haveshown that lysosomes in liver and other cell typesmay engage in digestion of the cell's own aged or

defective organelles, a process which has beentermed autophagy and which probably involvesthe formation of digestive vacuoles (secondarylysosomes) (11, 12). It was probable thatautophagy could occur in leukocytes, but the con-

ditions under which the process could be inducedwere unknown.

Ultrastructural studies of intracellular digestionin other cell types have shown that secondarylysosomes may take the form of residual bodiesconsisting of membrane-bounded whorls of lamel-lar material (myelin figures). Some of the cir-culating neutrophils in peripheral blood from sar-

coid patients who were treated with chloroquineshowed myelin figures. From the observations on

peripheral blood alone the possibility existed thatthe abnormal cytoplasmic granules were secondarylysosomes, perhaps representing the end stage ofdigestion of endogenous or exogenous material.

A study of the bone marrow leukocytes froma patient with abnormal cytoplasmic granules inmature, circulating neutrophils revealed that ultra-structural changes occur in cytoplasmic granulesof immature as well as mature cells. In neu-

trophilic myelocytes the abnormal granules appear

to be forming, for there are visible vacuolar, lamel-lar, and particulate components which are incor-porated into cytoplasmic granules; mature neu-

trophils contain myelin figures.On the basis of the findings in peripheral blood

and bone marrow there are three possible cellmechanisms alone or in combination which couldproduce the abnormal changes: phagocytosis, an

abnormality in cytoplasmic granule formation, orautophagy. The cell process which contributes tothe change is likely one to which the myelocyte ismost susceptible. In that case, phagocytosis couldbe excluded as the responsible process, since myelo-cytes are known not to engage in phagocytosis ofexogenous material (13). On the other hand, thechanges seen in early myelocytes are compatiblewith an effect of the drug on cytoplasmic granuleformation, but no direct evidence for such aneffect is available (14). The observed changescould also arise by autophagy, perhaps occurringin response to a damaging effect of the drug onsome cytoplasmic component.

In other words, the abnormal cytoplasmic gran-ules in myelocytes and mature neutrophils mayrepresent either primary lysosomes arising fromdefective granule formation or secondary lysosomesresulting from an autophagic process. A mean-ingful answer could be obtained only if a recog-nizable organelle, such as mitochondrion, wasfound within vacuoles observed in affected myelo-cytes. Autophagy then could be clearly implicatedas the process involved. In the specimens ex-amined there were no recognizable cytoplasmicorganelles within abnormal vacuoles or granules.In the case of the neutrophil, electron microscopichistochemistry for acid phosphatase is of little helpin distinguishing between primary and secondarylysosomes.

Eosinophils from certain patients treated withchloroquine also show structural abnormalities inimmature cells. Many cytoplasmic granules invery early myelocytes are normal; abnormal cyto-plasmic granules become prominent as the cellsmature. The more mature cells contain a markedreduction in content of the characteristics, normalcytoplasmic granule with an internal crystalloid.Instead, the cytoplasm is filled with vacuoles whichcontain amorphous material. The observed changesmay represent an arrest in granule formation oralteration of normal cytoplasmic granules afterthey are formed.

Normal lymphocytes are known not to take upexogenous material by either phagocytosis or pino-cytosis. In addition, these cells normally containfew if any cytoplasmic granules. Histochemistryat the ultrastructural level helped to establish thatthe myelin figures which may appear in lympho-

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MARTHAFEDORKO

cytes of persons given chloroquine were positivefor acid phosphatase. Autophagy is then the mostreasonable explanation for the origin of these ab-normal structures.

The influence of these ultrastructural changesin cytoplasmic granules on cell function is un-known. Clinically the patients were asymptomatic;they did not report intercurrent infections, andhealing from skin biopsy sites was unimpaired.Approximately one-half of the patients treatedwith chloroquine phosphate did not show changesin their leukocytes; the reason for this is not ap-parent at the present time.

The morphologic changes produced by chloro-quine in leukocytes and secretory cells of otherspecies as well as the characteristics of the chloro-quine effect with in vitro cell systems will be de-scribed in subsequent communications. The ultra-structural changes described here characterize oneform of acquired abnormal ("toxic") leukocytegranulation. The ultrastructure of other forms ofacquired leukocyte granulation is unknown. Thecell mechanisms responsible for abnormal leuko-cyte granulations including those in such heredi-tary disorders as the Chediak-Higashi syndromeand Alders anomaly remain to be investigated.

References

1. Cohn, Z. A., and J. G. Hirsch. 1960. The isolationand properties of the specific cytoplasmic granulesof rabbit polymorphonuclear leucocytes. J. Exptl.Med. 112: 983.

2. Hirsch, J. G., and Z. A. Cohn. 1960. Degranula-tion of polymorphonuclear leucocytes following

phagocytosis of microorganisms. J. Exptl. Med.112: 1005.

3. Biumer, A., H. Pau, and H. Conrads. 1959. Dar-stellung von Resochin bzw. Resochinderivaten inGeweben. Zeitschrift fur Rheumaforschung. 18:433.

4. Luft, J. 1961. Improvements in epoxy resin embed-ding methods. J. Biophys. Biochem. Cytol. 9: 409.

5. Venable, J., and R. Coggeshall. 1965. A simplifiedlead citrate stain for use in electron microscopy.J. Cell Biol. 25: 407.

6. Miller, F., E. de Harven, and G. E. Palade. 1966.The structure of eosinophil leucocyte granules inrodents and in man. J. Cell Biol. 31: 349.

7. Bryant, B. J., and L. S. Kelly. 1958. Autoradio-graphic studies of leucocyte formation. Proc. Soc.Exptl. Biol. Med. 99: 681.

8. Foot, E. C. 1965. Eosinophil turnover in the normalrat. Brit. J. Haematol. 11: 439.

9. Schneider, J., A. Nenna, J. Couture. 1963. Etudecomparative de la circulation dans le sang et del'elimination urinaire de la chloroquine base et dusulfate de chloroquine. Bull. World Health Organ.29: 417.

10. Visfeldt, J. 1964. Possible survival for years oflymphocytes in vitro without mitotic division.Acta. Pathol. Microbiol. Scand. 61: 319.

11. Ashford, T. P., and K. R. Porter. 1962. Cytoplasmiccomponents in hepatic cell lysosomes. J. Cell Biol.12: 198.

12. Swift, H., and Z. Hruban. 1964. Focal degradationas a biological process. Federation Proc. 23: 1026.

13. Jersild, M. 1948. Phagocytic activities of varioustypes of leucocytes. Acta Med. Scand., Suppl. 131:238.

14. Fedorko, M. F., and J. G. Hirsch. 1966. Cyto-plasmic granule formation in myelocytes. An elec-tron microscope radioautographic study on themechanism of formation of cytoplasmic granulesin rabbit heterophilic myelocytes. J. Cell. Biol.29: 307.

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