Reversible Phagocytosis in Rabbit Polymorphonuclear

Leukocytes

R. D. BERLIN, J. P. FERA, and J. R. PFEIFFER, Department of Physiology, University ofConnecticut Health Center, Farmington, Connecticut 06032

A B S T RAC T We have studied the fate of inertphagocytized particles in rabbit neutrophils. Neutro-phils release significant quantities of preingested oilemulsion. Roughly 50% of an ingested load is releasedin 40 min at 37°C. By electron microscopy the processof release appears to be by exocytosis: particles appearextruded through a network of processes oftenaccompanied by membranous vesicles. Exocytosis istemperature and glucose dependent but unlikephagocytosis does not require divalent cations. FromCoulter counter measurements virtually the entire cellpopulation appears to undergo the phagocytosis-exocytosis sequence. Neutrophils undergoing exocy-tosis remain intact as determined by direct counts,electron microscopy, and absence of lactate dehydrog-enase release. Moreover, by sequentially feedingdifferently labeled particles, it is shown that theprocesses of phagocytosis and exocytosis can occurconcurrently. Indeed, it is found that ingestionaccelerates release. The implications of these phe-nomena for membrane recycling, lysosom-al enzymerelease, and the killing of microorganisms arebriefly discussed.

INTRODUCTION

Recent studies of the fate of substances ingested bypinocytosis or phagocytosis have emphasized theircomplete degradation or retention within lysosomes asso-called residual bodies (1). In neutrophils andmacrophages it is usually assumed that ingestedparticulates are sequestered until the dissolution ofthe cell. On the other hand, the presence of "excretoryvacuoles" is well known in unicellular organisms.Exocytosis of latex beads during encystment ofamoebae has been well documented, although theprocess appears to require many hours (2).

We report here that rabbit neutrophils can releasea significant fraction of phagocytized oil emulsion. The

Received for publication 26 October 1978 and in revisedform 15 February 1979.

time-course of this release is similar to that of uptake.The release appears to occur by a process of reverseendocytosis (exocytosis). Cells undergoing releaseremain intact as determined by direct couints, absenceof lactate dehydrogenase release, and electroni micro-scopic examination. By sequentially feeding differentlylabeled particles, it is shown that the processes ofphagocytosis and "reverse phagocytosis" can occurconcurrently. Moreover, ingestion accelerates exocytosis.

METHODS

Cells. Rabbit polymorphonuclear leukocytes (PMN)1 wereobtained from glycogen-induced peritonieal exudates asbefore (3).

Medium. The basic medium consisted of 140( mMNaCl10 mMKCl, 10 mMHepes, pH 7.5. Wheni present the mediumconsists of: MgCl2, 2 mM; CaCI2, ).01 mM; glucose,5 mM; EDTA, 4 mM.

Phagocytosis and quantitation of particle content. Particlesof paraffin oil stabilized with albumin were prepared bysonication. Some preparations were labeled with oil red 0(ORO) as originally described l)y Stossel et al. (4). Inaddition, for some experiments the emiiulsioni was labeled withthe fluorescent lipophilic dye, perylenie. Like ORO, peryleneis not removed from the emulsion by repeated washing witha(lueous solvents. Cells at 107/ml were allowed to phagocytizeoil at a final concentrationi of 1.25% by volume. This wasnonsaturating so that uptake could be made nearly linear forup to 20 min. To qluantify oil content, the cells were firstcentrifuged at 40C for 6 mmin at 200 g. The superniate wassaved where appropriate for further ancalyses. The tubeabove the cell pellet was carefully wiped clean aind thepellet extracted into water-saturated n-butanol. After extraction,OROwas measured spectrophotomiietrically at 524 nim aindperylene by fluorescence using 436 aind 474 nm as excitationand emission wavelengths respectively. Unider the coniditionsemployed (dilute solutions with ORO of OD-0.1) therelation of absorption and fluorescence to dye conieentrationwas linear, and the two substancesi mixed could bemeasured independently. The amounit of emiiulsioni associatedwith the dye was callculated froIml ai palralffinl oil stock: 1 t.loil in 1 ml butanol saturated with watter corresponde(d to a0.29-OD at 524 nm. The perylene was dissolved in oil at

I Abbreviations used in this paper: ORO, oil redl 0; PMIN,rabbit polymorphonuclear leukocyte(s).

J. Clin. Invest. (© The American Society for Clinical Investigation, Inc. 0021-9738/79/06/1137/08 $1.00 1137Volume 63 June 1979 1137-1144

0.1 mM. The sensitivity of the detector was adjusted so that1 ,ul corresponded to 0.5 U of fluorescence intensity.

Coulter counter measurements of cell volume. A distribu-tion of "volumes" was recorded in a pulse-height analyzer(Channelyzer). The Coulter counter (Coulter ElectronicsInc., Hialeah, Fla.) was set with a T window of 10-100. Forthe Channelyzer, the base channel threshold was 10 and thewindow width 100. From the recorded data a relative volumecan be determined. However, the absolute volume dependson geometrical values that could change with phagocytosisor its reversal. Hence, the primary value of these data was toascertain whether the entire population or only a subfractionwas involved in the uptake and reversal phenomena. Theuse of the Coulter counter for assay of phagocytosis with wholeyeast cell has been recently reported (5), but the volumechanges obtained are complicated in the presence of particlesthat maybe absorbed to the surface. Absorption of oil emulsionas indicated (4) was found negligible by direct light micro-scopic examination.

Electron microscopy. Rabbit PMNwere fixed as a loosepellet in 1% glutaraldehyde in 0.1 M sodium cacodylate,pH 7.4, at room temperature for 1 h, rinsed in cacodylate, andpostfixed in 2%OS04 in cacodylate, pH 7.4, also at room tem-perature, for 1 h. The samples were dehydrated in gradedethanols and transferred through propylene oxide to Epon(Shell Chemical Co., Houston, Tex.) for embedding at 600Cfor 2 h. Uranyl acetate was avoided before embedding as itvariably extracted cell glycogen. Thin sections (silver) werecut and picked up on 300-mesh copper grids, doubly stainedwith uranyl acetate and lead citrate and examined on a Philips300 electron microscope (Philips Electronic Instruments, Inc.,Mahwah, N. J.). Approximately 1 gm thick sections werestained with 0.5% methylene blue, 0.5% Azure II in 0.5%borate, pH 7.4.

Enzyme assays. Lactate dehydrogenase was measured in0.1% Triton (Rohm and Hass Co., Philadelphia, Pa.) extractsof cell pellets or in supemates. Samples were excited at 340 nmand NADHoxidation was followed by the decrease in fluores-cence at 467 nm in the presence of excess pyruvate.

RESULTS

Phagocytosis. With 1.25% vol/vol emulsion in ourstandard medium, phagocytosis was linear for _20min when expressed as oil uptake per cell. It is im-portant that the cells separated from the emulsion bycentrifugation not be washed because this was found tolead to progressive losses of the number of cells pel-letable. Presumably this loss results from the flotationof relatively more engorged, buoyant cells. Thus, thevolume of oil ingested per 107 cells decreases progres-sively with washing. To control for any noningestedcontaminating emulsion in the centrifuged cell pellet,the oil content of a pellet obtained immediately aftermixing was determined; this amounted to <10% ofthe total in all samples and was usually <5%. Thisvalue was subtracted from the total to give the net in-ternalized oil. The amount of oil taken up during the20-min period of phagocytosis averaged 0.6 ,ul/107 cellsbut there was great variability from day-to-day (0.31-1.39 1.d/107 cells). The average value corresponds to-20% of the cell volume of 3.5 u/107 cells (6).

Reversal of phagocytosis. When the postphago-

cytic cells are suspended in medium without calciumor magnesium at 37°C a progressive decrease in pel-letable oil is observed. Fig. 1 shows average values±2 SDof 14 experiments. The oil associated with cellsis expressed as the fraction of the uptake attained inthe initial 20-min period of phagocytosis. In all experi-ments the number of cells pelletable after the post-phagocytic incubation period was determined. In ex-periments in which cell losses of > 10%were found, theresults were discarded. Note that the loss of emulsion-laden buoyant cells cannot account for the progressiveloss of emulsion with time. The release of lactate de-hydrogenase into the medium during the period ofreversal was also negligible, accounting for an averageof 3±2% (2 SE, seven experiments) of total cellularlactate dehydrogenase.

Factors that influence reversal. No reversal occursat 4°C (five experiments). The rate of reversal is re-duced =70% at room temperature (five experiments).

Unlike phagocytosis itself, reversal does not requireexogenous divalent cations. After 20 min incubationpostphagocytosis in medium containing 4 mMEDTA,the fraction of cell-associated oil remaining was0.59±0.076 (2 SE, eight experiments). This is actuallysomewhat less than the fraction obtained withoutEDTA, (0.68; Fig. 1).

Reversal is dependent on exogenous glucose. Inthese experiments cells were first allowed to phago-cytize in the presence of only 1 mMglucose withthe object of partially depleting endogenous glucosestores, although this was not shown directly. The cellswere then pelleted and resuspended in medium con-taining either no or 5 mMglucose. As shown in Fig. 2,little reversal is seen in the absence of glucose.

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FIGURE 1 Residual oil content vs. time of incubation inemulsion-free medium. The data are expressed as the fractionof oil content remaining after a period of loading by phagocy-tosis. The latter period was 10-20 min and was adjusted, usingdirect microscopic examination, so as to prevent overengorge-ment and thereby flotation and loss of cells. The middle lineis the average of 14 experiments, and the bars +2 SE. Theouter lines show the two most extreme experiments.

1138 R. D. Berlin, J. P. Fera, and J. R. Pfeiffer

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FIGURE 2 A representative experiment indicating the oilcontent in microliter per 107 cells vs. time postphagocytosisin emulsion-free medium without or with 5 mMglucose.Phagocytosis proceeded in 1 mMglucose.

CONTROLINC. 20'

CONTROL+EDTA, 20'+ 20'

PHAGOCYTOSIS

PHAGOCYTOSIS- EDTA

Little or no phagocytosis occurs in the presence ofEDTA (7). Thus, reversal in the absence of divalentcations establishes that reversal can occur under condi-tions that are not compatible with phagocytosis. Fromthe temperature and glucose dependencies it is prob-able that reversal is an active, energy-dependentprocess. In addition, these requirements establish thatthe decrease in oil content is not an artifact of the cen-trifugation procedures that are identical under all con-ditions.

Cell volume studies by Coulter counter. In allreversal experiments a progressive decrease in ratewith time was observed. In most cases the cell con-tent appeared to plateau. Weattempted to determineif a subpopulation of cells did not undergo reversalor if reversal slowed by some other mechanism. Coultercounter analysis showed that statistically, the entirepopulation underwent the ingestion-exocytosis se-quence. Fig. 3 illustrates pulse-height distributioncurves in an experiment that shows the populationdistribution of cell volume in control cells, its in-crease with phagocytosis, and its subsequent decreaseafter incubation (in this case with EDTA). Based on theposition of the peak height and assuming similar geo-metric factors at all stages, the relative increase involume after phagocytosis is m20%and this is halvedafter the reversal period. These changes are consistentwith the volume changes predicted on the basis ofmeasurements of cell-associated oil. More importantly,it is seen from the graph that there is a major shift inthe entire distribution curve towards higher volumewith phagocytosis, and lower with subsequent incuba-tion. The distribution curve is nearly symmetric andshows no indication of bimodality. These findingsindicate that the bulk of phagocytes show reversal.

Electron microscopic observations. It is well knownthat the resting neutrophil is highly granular and showsa subcortical region that excludes organelles (Fig. 4A).Phagocytic cells show characteristic pseudopods or

CELL VOLUME

FIGURE 3 A tracing of the Channelyzer recording of relativecell number vs. increasing cell volume. A total of 10,000 cellswere counted. Cells in complete medium incubated 20 min inparallel with phagocytic cells are shown in the left-hand peak.After 20 min, phagocytosis the distribution is shifted signifi-cantly to the right (larger volume) in a unimodal somewhatbroadened peak. On incubation for an additional 20-minperiod in medium with 4 mMEDTA, control cells are un-changed in distribution. Postphagocytic cells, however, arereduced in volume toward the control, again in a relatively nar-row unimodal peak. In this experiment there was no cell lossdetermined by direct counts under any condition.

areas surrounding the oil particles that exclude gran-ules initially (Fig. 4B). Degranulation into phagocyticvacuoles is frequently observed. Examination ofneutrophils after 20 min of phagocytosis in thick (0.5,.tm) sections, which allow distinction between intra-cellular and adsorbed particles, Fig. 5, reveals thatessentially all cells contain some oil particles. More-over, the bulk of ingested particles is completely inter-nalized.

After incubation in medium without emulsion, withor without EDTA, numerous cell profiles are seendepleted of granules (Fig. 6A). Oil particles are seen be-ing discharged into the medium. The particles are par-tially enveloped by granule-free cytoplasm. At whatmay be assumed are early stages of discharge, the cellperiphery is typically broken up into thin processes asthough fenestrated by numerous small perforations(6B and C). The particle is not surrounded by cell mem-brane. Numerous vesicular membranous structures areoften attached to the cell at regions of disgorgement.These morphological observations are strikinglysimilar to those of Stewart and Weisman (2), who de-scribed the exocytosis of plastic beads from Acantha-moeba preceding their encystment.

Simultaneous phagocytosis and exocytosis. InAmoebae, exocytosis occurs during a period in which

Reversible Phagocytosis 1139

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the capacity for phagocytosis is lost. We found thatthis is not the case for rabbit neutrophils.

In these experiments oil particles undergoing phago-cytosis or exocytosis were separately identified bybeing labeled with either OROor perylene. As noted,these labels could be quantified when mixed in butanolextracts. After 10 min phagocytosis of emulsion con-taining one label, the cells were centrifuged and al-lowed to phagocytize emulsion containing the second.At frequent intervals the cells were sampled for theircontents of both labels. Fig. 7 shows the result of suchan experiment, in which the initial phagocytosis was ofperylene-labeled emulsion. It is seen that the perylenecontent fell >50% in 15 min, whereas simultaneouslythe OROcontent increased linearly. There was no cellloss during the experiment. Changing the order inwhich the differently labeled emulsions was given,provided essentially the same result: loss of the firstlabel with increase of the second (five experiments).The rate of phagocytosis in the reversal period was notsignificantly different from that in the initial phase ofphagocytosis, although uptake during the secondperiod was usually not linear for the entire 15-mininterval.

Influence of phagocytosis on exocytosis. It ap-peared from the preceding experiments that the rate ofexocytosis was accelerated by concurrent phagocytosis.The fraction of the initial oil content remaining after10 min was 0.82 for exocytosis in medium with EDTAand 0.56 when phagocytosis occurred simultaneously(average of four experiments). This difference was sig-nificantatthe level ofP < 0.001. Because of the rapidityof this discharge and difficulties in preventing cell lossas a result of overloading of cells, we examined thecourse of reversal at 22°C with or without concurrentphagocytosis. Only experiments in which cell losseswere < 10%and lactate dehydrogenase <5%of the celltotal were considered.

Cells were first loaded with ORO-labeled oil byphagocytosis at 37°C. They were then gently cen-trifuged and resuspended at 22°C in either mediumcontaining glucose and EDTA(usual reversal medium)or in medium containing perylene-labeled emulsion.Incubation was then allowed to proceed at 22°C. Thetime-course (average of five experiments) illustrated inFig. 8 shows clearly the acceleration of exocytosisassociated with continued ingestion.

When cells are allowed to phagocytize two differ-ently labeled emulsions sequentially then suspendedin medium with EDTA and incubated, the rates ofloss of the two labels are nearly identical, indicatinglittle or no discrimination based on time of residence

within the cell. It must be emphasized however thatthe processing of ingested particles, indicate(d by lyso-somal frision with the phagocytic vesicle, occursrapidly (4).

DISCUSSION

The foregoing results establish the exocytosis ofpreviously phagocytized particles by rabbit PMN. Theextension of this phenomenon to other mammalian celltypes that are involved in inflammatory and immuneresponses is under study.

PMNrapidly disgorge roughly 50% of an ingestedload of inert particles without disruption as judgedfrom direct counts, absence of lactate dehydrogenaseexcretion, and their continued capacity for phagocyto-sis. Reversal of phagocytosis is glucose and tempera-ture dependent but does not require exogenous diva-lent cations. From pulse-height-volume analysis(Coulter counter), electron microscopy, and the extentof reversal, it is clear that the process takes place withinvirtually the entire PMNpopulation (i.e., it is not aproperty of a subclass of cells).

It is important, we feel, that cells studied for reversalof oil particle phagocytosis not be extensively washed.Controls show that a single centrifugation and carefulcleanup of emulsion adsorbed to the sides of the centri-fuge tube leaves a nonphagocytized residue of no>5-10% of the cell content of oil. Extensive washing in-stead simply removes an increasing fraction of the mostbuoyant, aggressive phagocytes. Without cell countsthis phenomenon is obscured by normalizing uptakedata to cell protein.

Although extensive quantitative studies of lysosomalenzyme release were not done, preliminary experi-ments show release of 8-glucuronidase and gluco-saminidase during reversal. Given the extensive fusionof lysosomes with phagocytic vacuoles containing oilparticles readily observable by electron microscopy(Fig. 6B) and previously documented by biochemicalanalysis (8), the exocytosis of particles must inevitablylead to excretion of lysosomal enzymes. Reversal con-stitutes still another mode in addition to "regurgita-tion during feeding" and "frustrated phagocytosis"by which lysosomal enzymes may also be dischargedinto the extracellular space. It should be emphasized,as shown in Fig. 5, that after cell loading most particlesare within the cell and cannot be simply lost throughdesorption during subsequent incubations. Moreover,as shown in Fig. 8, loss of previously ingested particlesis accelerated during the uptake of a second particle.Thus, conditions would appear to be favorable for the

FIGURE 4 Electron micrographs (- , 1 ,um). (A) Control, nonphagocytizing rabbit neutrophil.Abundant granules and peripheral exclusion zone are evident. (B) Phagocytizing rabbit neutrophil.Note familiar agranular pseudopod and lysosome-phagosome fusion (arrows).

FIGURE 5 A thick (_ I,ul) section of plastic embedded neutrophils aitfer 20 min phagocytosis.The section is stained with 0.5% methylene blue, 0.5% Azur II in borate, pH 7.4. Virtually all cellshave taken up oil dIroplets indicated by the clear areas i(ld most droplets atre fully interinalized.

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FIGURE 6 (A) Rabbit neutrophil undergoing exocytosis of previouisly inigested oil. Cell fixed after20 min incubation in EDTAmedium and removal of emiulsioni. Cytoplatsmi is depleted of graniules(compare Fig. 4A). The overlying cell appears broken uip by feniestrations anid various membranousvesicles and(or) osmiophilic (lysosomal?) mnaterial is loosely associatedi with the particle. (B and( C)The fenestrations are again visible. This is distinctly dlifferenit fromi the patterni of ingestioni (Fig.4B) in which there are smiooth pseudopodial extensionis that are niever initerrupted except f'or atsingle region that remains unenclosed (1). Note that where the cell is niot so (lepletecl of graniules,as in 4A, the region near the particle undergoing exocytosis is by conitratst dlevoid of graniules suig-gesting a filamentous network. Note also that the particle is niot suirroundedI by cell miembrane.

1142 R. D. Berlin, J. P. Fera, and J. R. Pfeiffer

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FIGURE 7 A plot of oil content vs. time. Cells were initiallyloaded by phagocytosis of perylene-labeled emulsion. Theywere then centrifuged, the supernate containing uningestedemulsion removed, and the cells resuspended in phagocyticmedium containing ORO-labeled emulsion. At intervals,aliquots were removed and the cells analyzed for their con-tents of perylene and ORO. It is seen that while there is aprogressive increase in ORO, perylene content falls by >50%in 15 min.

continued internalization of any incompletely en-veloped particles remaining after the first round ofphagocytosis. Reversal is, therefore, most likely theconsequence of some intracellular event.

The precise mechanism of exocytosis is not yet de-fined. The cell structure surrounding particles inprocess of exocytosis excludes ganules. Coupled withglucose and temperature dependencies it seems likelythat a filamentous contractile system is active in ex-pelling the particle, but this is not proven. A simiiilarargument was advanced by Stewart and Weisman (2) toexplain exocytosis of beads in Acanthamoebae. Al-though the population of amoebae took many hours toeliminate intracellular beads, it is possible that in-dividual cells could undergo exocytosis at rates com-parable to PMN. Again we note that inAcanthamoebae,this exocytosis occurs at a time during which phago-cytosis is blocked in contrast to the PMN, in whichphagocytosis and exocytosis can occur at the same time.

The possibility that membrane internalized withpinocytosis is recycled back to the plasma memnbranehas been inferred by Steinman et al. (9). Our studiessuggest the direct reinsertion of plasmiia membranecomponents during exocytosis: the release of particlesseems accompanied by fusion of the phagolysosomiiemembrane with the plasmalemmlla. It seems likely thatsome interiorized constituents of the latter are therebyrecycled. However, the presence of what appears to beshed membranous material near the site of particle

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FIGURE 8 Fraction of perylene-labeled phagocytized oilremaining after incubation at 22°C in medium containingEDTAor ORO-labeled emulsion. Cells were initially loadedby phagocytosis of perylene-labeled oil for 10 min at 37°C. Theplots are an average of five experiments in which cell loss andlactate dehydrogenase excretion were negligible. In bothcases loss of oil is evident, but the loss is clearly accelerated byphagocytosis. The difference between EDTAand phagocytosisincubations is significant at the P < 0.001 level. OROoil con-tent rose continuously by phagocytosis in the release period,and at 40 min (22°C) was roughly equal to that attained inthe initial 10 min ingestion (37°C) of perylene-labeled oil.

discharge suggests that significant regions of recycledmembrane are simply lost from the cell. In previousstudies of the fate of membrane constituents duringphagocytosis, we have shown in alveolar macrophagesthat membrane containing transport sites is not insertedfrom intracellular precursors during the period ofendocytosis (10), and argued for a similar lack inPMN. This is not contradicted by the present experi-ments since exocytosis would not effect significantchanges in membrane composition in the course of ashort period of phagocytosis, but rather should becomeimportant during prolonged recovery.

By use of different labels for the bovine serum al-bumin-coated emulsion it is shown that phagocytosisand exocytosis can occur simultaneously. Moreoveringestion clearly accelerates the rate of exocytosis.Whether this is signaled by a volume change per se orsome membrane event is not clear at this time. How-ever, the phenomenon has several interesting implica-tions. First, it is possible that cycling bacteria throughingestion and exocytosis could amplify the leukocyte'skilling potential. Thus, the number of organisms killedmight exceed the numuber present at any time withinthe cell. Second, feeding leukocytes an innocuousparticle could force the exocytosis of toxic ones in-cluding perhaps obligate intracellular microorganismsthat then would becomiie susceptible to extracellulartherapeutic agents. Realization of these possibilitiesmust await description of the range of particles andphagocytes that display reversal. However, our studieswith an inert nontoxic emulsion have made clear the

Reversible Phagocytosis 1143

existence of a rapid reversal process in mammalianneutrophils.

ACKNOWLEDGMENT

Supported by National Institutes of Health grants CA 15544and GM22621.

REFERENCES

1. Silverstein, S. C., R. M. Steinman, and Z. A. Cohn. 1977.Endocytosis. Ann. Rev. Biochem. 46: 669-722.

2. Stewart, J. R., and R. A. Weisman. 1972. Exocytosis oflatex beads during the encystment of Acanthamoeba.

J. Cell Biol. 52: 117-130.3. Kaiser, H. K., and W. D. Wood, Jr. 1962. Studies on the

pathogenesis of fever. IX. The production of endogenouspyrogen by polymorphonuclear leukocytes. J. Exp. Med.115: 27-35.

4. Stossel, T. P., R. J. Mason, J. Hartwig, and M. Vaughan.1972. Quantitative studies of phagocytosis by poly-morphonuclear leukocytes. Use of paraffin oil emulsions

to measure the iiitial rate of phagocytosis. J. Clin. In-vest. 51: 615-624.

5. Magnusson, K-E., C. Dahlgren, 0. Stendahl, and T. Sund-quist. 1977. Characteristics of the phagocytic processassessed by Coulter counter. Acta Pathol. Microbiol.Scand. Sect. C. Immunol. 85: 215-221.

6. Hawkins, R. A., and R. D. Berlin. 1969. Purine transportin polymorphonuclear leukocytes. Biochim. Biophys.Acta. 173: 324-337.

7. Berlin, R. D., and J. P. Fera. 1977. Changes in mem-brane microviscosity associated with phagocytosis: effectsof colchicine. Proc. Natl. Acad. Sci. U. S. A. 74: 1072-1076.

8. Stossel, T. P., J. D. Pollard, R. J. Mason, and M. Vaughan.1971. Isolation and properties of phagocytic vesicles frompolymorphonuclear leukocytes. J. Clin. Invest. 50:1745-1757.

9. Steinman, R. M., S. E. Brodie, and Z. A. Cohn. 1976.Membrane flow during pinocytosis. A stereologic analy-sis.J. Cell Biol. 68: 665-687.

10. Tsan, M. F., and R. D. Berlin. 1971. Effect of phagocy-tosis on membrane transport of nonelectrolytes. J. Exp.Med. 134: 1016-1035.

1144 R. D. Berlin, J. P. Fera, and J. R. Pfeiffer