LEUKOKINETIC STUDIES. III. THE DISTRIBUTION OF GRANULOCYTESIN THE BLOOD OF NORMAL SUBJECTS *

By J. W. ATHENS, S. 0. RAAB, 0. P. HAAB,t A. M. MAUER, H. ASHENBRUCKER,G. E. CARTWRIGHT AND M. M. WINTROBE

(From the Department of Medicine, University of Utah College of Medicine,Salt Lake City, Utah)

(Submitted for publication August 15, 1960; accepted September 14, 1960)

In a previous publication (1) it was noted thatwhen granulocytes were labeled in vitro with ra-dioactive diisopropylfluorophosphate (DFP32 ) andthen returned to the circulation of the donor, abouthalf of the labeled cells could not be found in thecirculation at the completion of the infusion (T0).Thereafter the remaining labeled cells left the cir-culation in a random fashion with a mean half-time disappearance (T.) of 6.6 hours.

Since cell damage and significant elution of thelabel could not be demonstrated tinder the condi-tions of the study, it was suggested that the im-mediate disappearance of half the infused cells wasdue to their rapid dilution in a larger pool thanthat calculated from the blood volume and the ve-nous granulocyte count.The concept that the circulating granulocyte

pool (CGP) does not constitute all of the intra-vascular leukocytes is not new. Vejlenis (2) re-viewed the lntumerotus inivestigations carried outup to 1938 and pointed out that as early as 1867Cohnheim observed white cells in a marginal posi-tion along the walls of venutles. By microscopicexamination it has since been demonstrated thatleukocytes may circulate freely in the blood, ad-here to the vascular enidothelium in sites wherethe blood flow is relatively slow and then onceagain re-enter the circulation in a process of con-tinuous exchange (2).

If DFP32-labeled granulocytes come into rapidequilibrium with marginated granulocytes, it mightbe expected that one could account for most if notall of the labeled cells bv mobilizing the marginatedcells into the circulation Nwith epinephrine. M\Io-

* This investigation was supported in part by researchgrants (C-2231 and CY 2349) from the National CancerInstitute and in part by a graduate training grant (2A-5098) from the National Institute of Arthritis andMetabolic Diseases, Bethesda, Md.

t Fellow of the Leukemia Society Inc., New York,N. Y.

bilization of the marginated cells into the circula-tion after equilibration has occurred should notalter the specific activity of the cells in the CGP.The purpose of this paper is to present evi-

dence that labeled granulocytes are distributed ina larger pool than the CGP and to present data onthe size of this distribution compartment in 45 nor-mal subj ects.

MATERIAL AND METHODS

Volunteers from the Utah State Prison were used inthese studies. All of the subjects were healthy coopera-tive males, 20 to 50 years of age, with normal leukocytevalues, who were free of even minor infections. Thesubjects were allowed to have breakfast at 6 a.m. Theinfusion of labeled blood was begun between 8 and 10 a.m.The methods for labeling granulocytes in vitro (1), for

the isolation of leukocytes from the blood samples andfor the determination of leukocyte radioactivity were de-scribed previously (3).

Total and differential leukocyte counts were made atthe beginning of an experiment and at appropriate timeintervals during the study. When reference is made inthe text to granulocyte pools, these terms refer to theneutrophilic granulocyte series. Eosinophils and baso-phils have not been included in the granulocyte total be-cause, by means of autoradiographic studies with tritiatedDFP, it has been found that these cells do not label (4).The number of granulocytes per millimeter cubed was

calculated from the total and the differential leukocytecounts. The blood volume of a subject was assumed tobe 2.68 L per m2 of body surface area (5). The sur-face area was estimated from the nomogram of Du-Bois (6). The circulating granulocyte pool was calcu-lated from the number of granulocytes per millimetercubed and the estimated blood volume.Three 20 ml samples of blood were removed from the

bag just prior to the infusion, and the specific activityof the isolated leukocytes, expressed as counts per min-ute per milligram of leukocyte nitrogen, was determined.The mean value for the 3 determinations was taken torepresent the specific activity of the granulocytes in thebag (SA of bag G). The concentration of granulocytesin the bag was calculated from the total and differen-tial leukocyte counts determined directly on the bag bloodafter incubation with DFP32. The volume of blood ad-

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ministered to the subject, usually between 450 and 500 ml,was calculated from the weight of blood injected and an

assumed specific gravity of 1.055. The total number oflabeled granulocytes given to the subject (no. G in bag)was then calculated.The blood was administered to the original donor over

a 15 to 20 minute period. Within 3 minutes after thecompletion of the infusion (T,), three 20 ml samples ofblood were withdrawn from the subject and the specificactivity of the granulocytes in the circulation was deter-mined. The mean value of the 3 determinations was

taken to represent the specific activity of the granulo-cytes in the blood at To (SA blood G).The percentage of infused cells present in the CGP at

To was calculated as follows:

Per cent of infused Gpresent in CGP at To

_ CGP X SA blood G 100no. G in bag X SA of bag G X

The number of cells in the total blood granulocyte pool(TBGP) was calculated as follows:

TBGP = bag X nio. in bag

SA of blood G

RESULTS

Influence of epinephrine and exercise on thepercentage of infused granulocytes present in thecirculation at To. In an attempt to account forthe labeled cells apparently lost from the circula-tion during the infusion, the following studieswere carried out. Blood from each of five subjectswas labeled with DFP32, infused into the donor,and the percentage of infused graulocytes presentin the circulation at To determined. Five dayslater the studies were repeated in the same sub-jects except that 0.5 mg of epinephrine was addedto the labeled blood just prior to infusion. Ineach epinephrine study as compared with thecontrol, there was an increase in granulocytecount at the end of the infusion ranging from 37to 179 per cent. In addition, the percentage of in-

fused granulocytes in the circulation increasedfrom a mean of 48.0 to a mean of 78.8. Neverthe-less, in no instance could all of the infused cells beaccounted for in the circulation (Table I).

Similar studies were carried out in four addi-tional subjects except that a 440 yard run at the

completion of the infusion was used to mobilizegranulocytes rather than epinephrine. After ex-

ercise the granulocyte count increased 64 per centon the average and the proportion of infused gran-ulocytes which could be accounted for in the cir-

TABLE I

Influence of epinephrine on the percentage of infusedgranulocytes present in the CGP at To *

Per cent infused Gin CGP at To

Subject Control Epineplhrine A Gt

11-164 47 88 1.7211-166 55 82 1.3711-168 52 81 1.4211-204 49 71 1.9911-208 37 72 2.79

Mean 48.0 78.8 1.86

* G = granulocytes; CGP = circulating granulocytepool; To = completion of infusion.

t Ratio of granulocyte count at To of epinephrine studyto that in the control study.

culation increased from an average of 45 to 79 percent. The results varied considerably. It is ofinterest that in two subjects (III-4 and III-10) 96and 100 per cent of the infused cells were foundin the circulation (Table II).

Influence of epinephrine and of exercise on thespecific activity of the granulocytes in the circula-tion. To assess the possibility of a continual ex-change of granulocytes between the CGP and themarginated cells, each of eight subjects was givenan infusion of autogenous, DFP32-labeled blood.Three hours after completion of the infusion, atwhich time the granulocyte disappearance curvewas well established, the concentration and thespecific activity of the granulocytes in the CGPwere determined. Immediately thereafter 0.4 mgepinephrine in 50 ml of isotonic saline was ad-ministered intravenously over a period of 4 to 27minutes. The concentration and the specific ac-tivity of the granulocytes were measured again.In two subjects the granulocyte count increased

TABLE II

Influence of exercise on the percentage of infusedgranulocytes present in the CGP at To

Per cent infused Gin CGP at To

Subject Control Exercise A G*

111-2 30 54 1.73111-4 43 101 2.38III-10 62 96 1.25111-12 55 65 1.18

Mean 45.0 79.0 1.64

* Ratio of granulocyte count at To of exercise study tothat in the control study.

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TABLE III

The effect of epinephrine and exercise ongranulocyte specific activity (SA) *

SA, cpm per mg N

Before After A Gt

Epinephrine infusiont 1,387 1,331 1.47Exercise (440 yard run)§ 697 756 1.23

* The epinephrine or exercise was administered 3 hoursafter infusion of the labeled blood.

t Ratio of granulocyte count after epinephrine injectionor exercise to that before.

$ Values are the mean of 6 subjects.§ Values are the mean of 2 subjects.

less than 10 per cent and these studies were, there-fore, discarded. In the other six subjects, themean granulocyte count increased 47 per cent andyet there was no significant change in the specificactivity of the granulocytes in the CGP (TableIII).A similar study was carried out in three sub-

jects except that a 440 yard run was used in placeof epinephrine. Neutrophilia was not observedin one subject. Therefore, this subject is not in-cluded in Table III. The mean granulocyte countin the other two subjects increased 23 per cent inabout 2 minutes with no significant change in thespecific activity of the granulocytes in the CGP.

Inability of epinephrine to mobilize damagedgranulocytes. To determine whether cells whichhave been deliberately damaged and presumablyhave been rapidly removed from the CGP couldbe mobilized back into the CGP by an infusion ofepinephrine, blood was withdrawn from four sub-jects, labeled with DFP32 and stored for 24 hoursat 4° C. Storage of blood has previously beenshown to result in the immediate disappearancefrom the CGP of over 90 per cent of the injected

TABLE IV

Failure of epinephrine to mobilizedamaged granulocytes

Per cent of infused Gpresent in CGP at To

Stored cells

Subject Stored cells epinephrine

I-119 9.3 9.91-121 5.3 5.91-153 8.0 6.81-155 3.9 5.4

Mean 6.6 7.0

cells (1). The cells were then infused into therespective donors and the fraction of the injectedcells present in the circulation at To was deter-mined. No untoward reactions occurred duringor after the infusions and the granulocyte countsremained unchanged.The studies were repeated in the same individu-

als at a later date, and 0.4 mg of epinephrine was

added to the blood in the bag. The results are pre-sented in Table IV. The damaged granulocyteswere rapidly removed from the circulation duringthe infusion and were not in the circulation at To,Furthermore, the epinephrine failed to mobilizethe damaged cells even though a mean increase ingranulocyte count of 23 per cent occurred.The size of the TBGP and CGP in 45 normal

subjects. Since the apparent loss of 50 per centof the DFP32-labeled granulocytes during the in-fusion appears to be due to their distribution in a

pool larger than the CGP and not due to cell dam-age, the size of this distribution compartment(TBGP) was measured in 45 normal subjects(Table V).The mean TBGP was 65.3 X 107 granulocytes

per kg of body weight and the mean CGP was

30.7 X 107 granulocytes per kg.Influence of epinephrine and of exercise on the

distribution of granulocytes within TBGP. Toascertain the effect of epinephrine on the size of theTBGP and the distribution of cells therein, bloodfrom each of eight subjects was labeled withDFP32, infused into the donor, and the TBGPand CGP were determined. Five days later thestudies were repeated in the same subjects exceptthat 0.5 mg epinephrine was added to the labeledblood. The size of the TBGP was unaffected bythe epinephrine (Table VI). However, the mean

number of granulocy-tes in the CGP increasedfrom 34.2 x 107 cells per kg to 50.5 x 107 cellsper kg, a 48 per cent increase after administrationof epinephrine.

TABLE V

The size of the total blood granulocyte pool (TBGP) andcirculating granulocyte pool (CGP) in 45 normal subjects

Mean ±1 SD Range

TBGP, cells X 107/kg 65.3 +L 22.7 36-117CGP, cells X 107/kg 30.7 i 11.8 15- 54TBGP-CGP, cells X 107/kg 34.6 i 15.6 12- 73

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TABLE VI

Influence of epinephrine and exercise on thesize of the TBGP and the CGP

Experimentalcondition TBGP CGP

X 10 7 G/kg XiO7G/kgControl study* 68.2 34.2

(46-105) (17-54)After epinephrine

infusion* 70.2 50.5(45-117) (31-70)

Control studyt 48.2 24.2(34- 56) (16-41)

After exerciset 47.1 38.6(36- 53) (28-51)

* These values are the mean of studies oln 8 subjects.t These values are the mean of studies on 3 subjects.

Similar studies were carried out in three addi-tional subjects except that a 440 yard run at thecompletion of the infusion was used to mobilizegranulocytes. Under the influence of exercisethe TBGP size was also unchanged and again thecells were shifted into the CGP to increase itssize by 60 per cent (Table VI).

DISCUSSION

The results of these studies indicate that labeledgranulocytes are distributed in a pool of cellswhich is larger than that calculated from the bloodvolume and from the concentration of granulo-cytes in the circulating blood. This dilution ac-counts, in part at least, for the previous olbserva-tion (1), confirmed herein, that only about 50 percent of the infused cells can be accounted for inthe CGP at the completion of aln infusion. Anaverage of 79 per cent of the infused, labeled cellswas present in the CGP following the administra-tion of epinephrine or after physical exercise.

Since only 71 to 88 per cent of the injected cellswas found in the CGP following epinephrine in-fusion (Table I) and 54 to 100 per cent after vigor-ous exercise (Table II), it may be argued that avariable proportion of the labeled cells was dam-aged irreversibly or that this proportion of thelabel eluted rapidly from the granulocytes. How-ever, in view of our previous studies relevant todamage and elution (3), it seems unlikely thateither damage or elution accounts for this imme-diate loss of cells from the circulation. A morereasonable explanation for our inability to account

for all of the injected cells in every experiment isthat epinephrine and exercise did not mobilize allof the granulocytes in each subject. Indeed, thisis suggested by the considerable variation of 0 to278 per cent in neutrophilic response from personto person.

Equilibration between the granulocytes in theCGP and in the "noncirculating pool" was bothrapid and complete. When the "noncirculatinggranulocytes" were mobilized into the CGP withinminutes by either epinephrine or exercise (TableIII), there was no change in the specific activityof the cells in the CGP. Therefore, these two poolsmay be considered as one pool kinetically, and thesize of this pool (TBGP) may be determined bythe use of the isotope dilution principle.

Measurement of the TBGP in normal subjectsrevealed that the mean TBGP is 2.1 times thesize of the CGP. Although direct quantitativemeasurements of the size of the total blood granu-locyte pool have not been made before, it was esti-mated that there are as many granulocytes in themarginal pool as in the circulating blood (7).

It is interesting that neither epinephrine nor ex-ercise influenced the size of the TBGP, althoughboth the drug and the physical exertion caused ashift of cells from the noncirculating pool to theCGP (Table VI). This type of alteration in thedistribution of granulocytes within the blood hasbeen the subject of speculation for many yearsand was called "pseudo-leukocytosis" by earlierinvestigators (2, 8). The present study providesthe first evidence for the existence of this type ofleukocytosis. One might speculate that the acuteneutrophilia which is associated with epileptiformseizures (9) and paroxysmal tachycardia (10)may also be of this type.Our studies do not provide evidence for the

anatomical location of the noncirculating pool ofgranulocytes. However, as indicated in the in-troduction, there is abundant evidence that thereis a continuous exchange of granulocytes betweenthe free-flowing axial blood and the more slowlymoving marginal stream (2). Vejlens (2)showed that when the velocity of blood flow isreduced, granulocytes tend to assume a marginalposition, become adherent to the walls of smallveins and are withdrawn from the axial stream.When the velocity of blood flow is increased,granulocytes enter the axial stream and are car-

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ried into the circulating blood. Since both exer-

cise and epinephrine increase blood flow, it isreasonable to assume that these stimuli producegranuloc)ytosis by mobilizing marginated cells.The work of Ambrus and Ambrus (11) and ofBierman and colleagues (12) suggests that thevascular system of the lungs is of particular sig-nificance in the margination of granulocytes. Theimportance of the vascular systemi of the splanchnicviscera (13) and extremities (14) has been dem-onstrated by the work of others. Apparently, thespleen plays a minor role in this regard, sincegranulocytosis induced by epinephrine is neitherabolished nor diminished in splenectomized sub-

jects (9).SUMMARY

1. When granulocytes are labeled in vitro withradioactive diisopropylfluorophosphate and re-

turned to the circulation of the donior, only about50 per cent of the labeled granulocytes is presentin the circulation at the completion of the infusion.It was postulated previously that the initial rapidegress of 50 per cent of the cells represented dis-tribution of the injected granulocytes in a largerpool than that calculated from the blood volumeand the venous granulocyte count. The experi-ments reported herein were designed to test thevalidity of this hypothesis. The following obser-vations were made. a) An average of 78.8 per

cent of the labeled granulocytes which were in-fused could be accounted for in the circulatingblood when epinephrine was given with the in-fused cells. b) An average of 79 per cent of thelabeled granulocytes was found in the circulationwhen the subjects exercised immediately followingthe completion of the infusion. c) Neither epi-nephrine nor exercise increased the size of thetotal distribution compartment (total blood granu-

locyte pool), although both the drug and physicalexertion increased the size of the circulating granu-

locyte pool. d) Granulocytes damaged by storagecould not be prevented from leaving the circulationby the administration of epinephrine. e) Whengranulocytes were mobilized into the circulation 3hours after the infusion of labeled granulocytes,there was no change in the specific activity of thecells in the circulation.

2. These studies are interpreted as indicatingthat infused granulocytes are distributed in a total

blood granulocyte pool (TBGP) made up of twocompartments which are in rapid equilibrium witheach other. These pools have been designated thecirculating granulocyte pool (CGP) and the mar-ginal granulocyte pool (MGP).

3. The size of these pools has been measured in45 normal male subjects. The mean values + 1SD, expressed as number of cells x 107 per kg ofbody weight, were as follows: TBGP, 65.3 ± 22.7;CGP, 30.7 ± 11.8; and MGP, 34.6 + 15.6.

ACKNOWLEDGMENTS

The cooperation of Dr. William Knott and WardenJohn Turner of the Utah State Prison is very much ap-preciated. We are also grateful to those inmates of theUtah State Prison who volunteered for these studies.Mrs. Roseann Moore and Joyce Rose, and Misses DorisKurth and Viktoria Wohlrab provided valuable techni-cal assistance. WVe are grateful to Dr. M. H. Kuizenga,Upjohn Co., Kalamazoo, Mich., for the supplies of he-Iarin sodium; to Dr. D. M. Monroe, Dow Corning,Corp., Los Angeles, Calif., for the silicone 4141; to Dr.(G. M. Karus, Film Dept., E. I. DuPont de Nemours andCo., \Vilmington, Del., for the Mylar; and to Dr. J. H.Brewer, Hynson, Westcott and Dunning, Baltimore, Md.,for the supply of crude cobra venom.

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