INFECTION AND IMMUNITY, Apr. 1970, p. 327-333Copyright © 1970 American Society for Microbiology

Vol. 1, No. 4Printed in U.S.A.

Function of the Reticuloendothelial SystemIV. Evidence for Two Types of Particle-Induced Reticuloendothelial Paralysis'

SIGURD J. NORMANN

Departmenzt of Pathology, University of Florida, Gainesville, Florida 32601

Received for publication 28 November 1969

Reticuloendothelial system (RES) phagocytosis has been quantitated afterintravenous injection of two different sets of particles by determining the clearancerate of subsequently injected identical or nonidentical particles. Injection of carbonproduced a biphasic RES paralysis consisting of an early transient phase followedby a delayed sustained phase. The two phases were separated by a distinct intervalof greatly augmented clearance rates. The injection of aggregated albumin was

followed only by a single period of depressed clearance, which corresponded to thefirst phase of carbon-induced inhibition. This first phase, designated immediate RESparalysis, was initiated by particle injection and its duration was related to the rateof particle removal, to the dose of particles injected, and to the presence of theparticles in the circulation. The second phase, designated delayed RES paralysis,began sometime after the particles had been engulfed by the cells, was independentof the rate of particle removal, and persisted without the presence of measurableparticles in the circulation. The evidence indicates that the immediate paralysisarises from a competition between the particles in the circulation, whereas thedelayed paralysis arises from a cellular derangement inhibitory to further phago-cytosis. In contrast to the usual description of RES blockade as a single sustainedperiod of depression, the present experiments indicate that the phenomenon has twophases which can be dissociated in time and mechanism.

The uptake of certain colloids by phagocyticcells in vivo can be inhibited by the prior injec-tion of the same or a different colloid. Frequently,inert particles are used to induce this depressedstate known as "reticuloendothelial (RES)blockade." Because such particles cannot beeliminated from the cell, it was thought that thedecreased clearance rates represented cellularsaturation and that the onset of the "blockade"dated from the progressive accumulation withinthe cells of the first set of particles injected (2, 3).Recently, Normann, Lagunoff, and Benditt (20)measured simultaneously the vascular clearanceof two dissimilar colloids (carbon and aggre-gated albumin), after their sequential injectioninto the circulation of a rat. The clearance rateof both particles was inhibited when both werepresent in the circulation. Their findings couldnot be explained on the basis of cellular satiationand suggested that one phase of RES paralysisis associated with the immediate events arisingfrom the presence of particles in the circulation.On the other hand, Parker and Finney (22) ob-

1 A preliminary report of the material presented here was madeat the 51st annual meeting of the Federations of AmericanSocieties for Experimental Biology, 1967.

served in mice that a period of normal clearancemay follow particle injection and exist prior tothe onset of RES paralysis. This latter observationsuggests that a second period of RES depressionmay follow particle iniection and represent someform of cellular derangement induced by colloidingestion. Therefore. it appears probable that theusual description of "RES blockade" as a singlesustained period of depression, originating withparticle injection, may not be correct; rather, thephenomenon may be composed of componentparts which may be dissociated in time andmechanism.The present report examines the possibility

that different phases of RES paralysis exist byinvestigating the behavior of the system immedi-ately after particle exposure in contrast to thebehavior evident 24 hr later. Two dissimilar col-loids were studied because the response of theRES to inert colloids such as carbon may bedifferent from the response to metabolizablecolloids such as aggregated albumin. The experi-ments demonstrate that two periods of reducedclearance rates are associated with "RES block-ade" and that the immediate paralysis initiatedby particle injection is a decidedly different event

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NORMANN

than the delayed paralysis manifested severalhours later.

MATERIALS AND METHODSMale Sprague-Dawley rats weighing between 150

and 250 g were used after anesthesia with intraperi-toneal pentobarbital sodium. The carbon suspensionmanufactured by Gunther-Wagner lot C-11-1431awas obtained from John Henschel and Co., NewYork, and used as supplied. Gelatin powder derivedfrom swine skin was obtained from Sigma ChemicalCo. (St. Louis, Mo.). Gelatin for injection was pre-pared as a 3% solution in 0.15 M saline. In some ex-periments, gelatin was added to the carbon prepara-tion prior to its injection into animals by the method ofBiozzi, Benacerraf, and Halpern (2). However, thetotal amount of gelatin added to carbon was held con-stant so that only 5 mg of gelatin per 100 g of bodyweight was injected into any animal irrespective of thecarbon dose. Aggregated albumin was prepared bycontrolled heating of bovine serum albumin and waslabeled with the fluorescent reagent dimethylamino-napthelene suphonyl chloride (DNS-Cl from SigmaChemical Co.) as described previously (20).The vascular clearance of carbon or DNS-labeled

aggregated albumin was determined by injecting in-travenously a standard dose of either 10 mg of carbonor 4 mg of aggregated albumin per 100 g. After injec-tion, blood samples were obtained from the femoralvein at regular time intervals. Clearance was measuredover a 15-min period except in those instances of ac-celerated clearance. Blood was obtained in heparin-washed syringes with particular care taken not to in-troduce the anticoagulant into the circulation. Thecarbon concentration in each sample was determinedby optical density measurement (20), where as theclearance of the aggregated albumin was measured bythe decrease in plasma fluorescence (20). The plot ofthe logarithm of the particle concentration versus timeyielded a straight line for both particle suspensions;the slope of this line was computed by linear regressionand designated the clearance rate constant or K. Com-parisons between the clearance rates obtained indifferent experiments was performed using Student's ttest for comparison between group means with un-equal population sizes (21).

In the studies to be described, the initial particleinjection was followed by a subsequent injection ofeithercarbonorlabeled aggregated albumin. The timeinterval between the two injections was varied so thatthe effect induced by the initial injection could beevaluated at different times. Multiple determinationswere made at each interval, and the value recordedrepresents an average determination on two or moreanimals.

RESULTSImmediate RES paralysis. A typical curve rep-

resenting the different rates of carbon clearanceobserved after injection of various doses of ag-gregated albumin is shown in Fig. 1. When thecarbon was administered shortly after the albu-min, a marked reduction in carbon clearance

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Aggregated T ilE IN HOURSAlbu mi n

FIG. 1. Effect of aggregated albumin injection onthe clearance rate of subsequently administered carbon.The dose of aggregated albumin injected is indicated atthe top of each carbon line. The 95% confidenice limitfor the normal rate of carbon removal was K = 0.033to K = 0.039.

rate was observed for each dose. However, therate of carbon removal was relatively constantand independent of the amount of albumin in-jected (K = 0.015 compared to a control of 0.036with a standard deviation of 0.009 determined on50 animals). A more effective paralysis of RESfunction as measured by carbon was not achievedby increasing the dose of aggregated albumin. Onthe other hand, the duration of the paralysis wasdependent on the albumin dose and lengthened asthe dose of the particle injected increased. Foraggregated albumin doses greater than 4 mg/100g of body weight, the paralysis terminated in astate of accelerated clearance (K greater than0.060). Induction of the accelerated clearancestate was not as effective nor was the duration sus-tained when the dose was reduced to 1 mg/100 gand was barely apparent at a dose of 0.5 mg/100g. Moreover, the accelerated clearance state in-duced by a 10-mg dose had disappeared by 24 hr(Table 1), whereas that induced with a 30-mgdose produced significant elevation in clearancerate at that time. Thus, it appeared that the in-duction and the duration of the increased carbonremoval rate depended upon the dose of aggre-gated albumin injected.

In the next series of experiments, the sequenceof particle injection was reversed and the effectof an initial injection of carbon was evaluated bythe clearance of a subsequent injection of labeledaggregated albumin (Fig. 2). Three different in-jections of carbon were made of 10, 20, and 30mg/100 g. When the aggregated albumin was in-jected immediately after the carbon and essentiallyduring its removal, the rate of albumin clearance(K = 0.004) was significantly less than the rateobserved in the absence of carbon (control albu-min clearance K = 0.008 with standard deviation

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FUNCTION OF RETICULOENDOTHELIAL SYSTEM. IV

TABLE 1. Effect of carbonz or aggregated albumin inijectioni onl the clearalnce rate of asubsequentt injectiont of carbon (10 mg/100 g)

4Dose initial Avg clearance rate K of a subsequent carbon injectionaInitial injection injection

(mg/lO00 g) 6 24

Aggregated albumin 10 0.017 + 0.007c 0.078 ± 0.020 0.040 i 0.01830 0.017 i 0.005c 0.017 4 0 007c 0.060 i 0.013

Carbon 10 0.020 i 0.009c 0.036 ± 0.007 0.054 i 0.02030 0.017 + 0.004c 0.083 ± 0.030 0.020 ± 0.009c

a Control: K = 0.036 ± 0.009 (50 determinations). Each value recorded represents the mean ± 1 stand-ard deviation determined on 10 animals. Two periods of clearance depression are evident for large carbondoses, but only a single period for small carbon doses. Aggregated albumin regardless of dose producesonly one period of depression.

b Hours after initial injection.c Value significantly less than control (P < 0.01).

60 75TINIE IN rMINUTESCarbon

FIG. 2. Effect of carboni injectionz onl the clearancerate of subsequenitly injected aggregated albumin. The95% confidence limit for the normal rate ofaggregatedalbumin removal was K = 0.0074 to K = 0.0087.

of 0.001 determined on 10 animals). Eventually,the rate of albumin clearance returned to normaland, in some instances, actually exceeded thenormal clearance value. A longer period of de-pressed albumin clearance was observed with thelarger carbon doses.

This depressant effect of carbon on RES phago-cytosis was also evaluated by the clearance of asubsequent injection of the same particle. Thissequence of particles was chosen because carbonis frequently used to induce "RES blockade," andthe extent of the paralysis induced by carbon (andother particles) is often evaluated by a subsequentcarbon injection. However, very early time in-tervals could not be evaluated due to residualcarbon in the circulation. Accordingly, the earliesttime at which the second carbon injection wasmade depended upon the removal rate of thefirst carbon dose and was given when 10% or lessof the initial carbon remained in the circulation(generally 0.5 hr for 10 mg and 1 hr for 30 mg/100

.090-

080-

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o-O 10mg Carbon / 100gmA^ ^ 30mg Carbon 100gm

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TI tE IN HOURS AFTER INJECTION

FIG. 3. Effect of carbon injection on the clearancerate of a subsequent injection of a standardized dose ofcarbon. The 95% confidence limit for the normal rateof carbonz removal was K = 0.033 to K = 0.039.

g). Under these conditions, a second carbon in-jection was cleared at a rate slower than that ob-served when the same dose of carbon was givenalone (Fig. 3). At 2 hr after the initial carbon in-jection, the rate of clearance after a 10-mg doseaveraged 0.040, a value greater than the control(K = 0.036), although not significantly different.

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INFEC. IMMUN.

At the same time period, the clearance rate afteran initial injection of 30 mg of carbon per 100 gwas still significantly retarded (P < 0.05), averag-ing K = 0.030. However, the clearance rate wasnow recovering rapidly for, at 3 hr, greatly aug-mented clearance rates were observed. This ele-vated clearance was found after injection of 30mg of carbon but not 10 mg of carbon per 100 g.Thus depressed clearance rates, as measured bycarbon, did follow prior carbon injection, and theduration of this depression appeared to be longerwith larger carbon doses.The above experiments showed that an initial

injection of either carbon or aggregated albumindelays the vascular clearance of a subsequentcolloid injection, provided the second colloid isinjected shortly after the first and essentiallyduring its clearance from the circulation. Thislatter fact can be deduced from the observationsthat closely spaced injections of similar and dis-similar colloids produce inhibition, from thecalculated clearances of each colloid (dc/dt =

-2.3 Kt where c is concentration and t is time),and from the observation that inhibition inducedby carbon lasts but a short time beyond the pointof detectable carbon in the circulation.Delayed RES paralysis. Table 1 presents data

comparing the immediate and delayed responsesof the RES to injection of either carbon or ag-gregated albumin. Both a large (30 mg/100 g) anda small (10 mg/100 g) dose of each colloid were

examined. Whereas the aggregated albumin pro-duced only a single period of depressed clearance,two periods of inhibited clearance clearly existedfor the larger carbon dose. The first period oc-curred early, coincident with particle injection,and lasted only a short time. The second periodoccurred late and was evident at 24 hr. The twoperiods of retarded clearance were clearly sepa-rated by an interval of accelerated clearance, asevident by the values reported for 6 hr. In con-trast, only the first period of depression was found

for the smaller carbon dose, as there was no de-pression of clearance rate at 24 hr; in fact, theclearance rate was actually greater than normal.Thus, a second period of clearance rate depressioncan occur after large doses of carbon, and the on-set of the depression begins some time after theparticles from the initial injection have beencleared from the circulation.

Effect of gelatin injection: a frequently usedparticle-stabilizing agent. Whereas aggregatedalbumin needs no stabilizing agent, carbon mustbe stabilized to prevent its flocculation in blood.Although the carbon preparation supplied byGunther-Wagner is stabilized with partially hy-drolyzed fish gelatin, many investigators-in-cluding Biozzi, Benacerraf, and Halpern (2),who developed the carbon clearance method ofevaluating RES function-increase the carriercolloid presumably to render the suspension morestable in blood. It appears probable that the con-tent of gelatin and its source might be importantvariables. Accordingly, gelatin alone and gelatinaddition to the carbon preparation were ex-

amined for their effect on carbon removal andinduction of "RES blockade."When gelatin was added to the carbon prepara-

tion in vitro and prior to its injection into animals,the carbon clearance rate decreased. This effecthas been previously observed (10, 17). In thepresent experiments, the same effect could also beachieved by the injection of gelatin alone. Table 2compares the effect upon carbon removal rate ofa prior injection of gelatin alone (5 mg/100 g),of carbon alone without supplemental gelatin,and of carbon with supplemental gelatin. Thefollowing features emerged. First, the gelatinalone was sufficient to produce a prolonged de-pression in the clearance rate of subsequently in-jected carbon. This effect occurred coincident tothe gelatin injection and persisted for up to 24 hr.Secondly, if gelatin were added to the carbonpreparation in vitro and prior to its injection into

TABLE 2. Effect of gelatin on the clearance rate of subsequently injected carbon

Interval Avg rate of clearance of a 2nd injection of 10 mg/100 g of carbon, after a 1st injection per 100 g ofabetween1st and2nd 30mg of carbon + 10 mg of carbon +injection 5 mg of gelatin 30 mg of carbon 5 mg of gelatin 5 mg of gelatin

hr

2 0.020 i 0.006 0.030 :1: 0.007 0.017 ± 0.005 0.019 i 0.0044 0.019 i 0.007 0.085 i 0.022 0.014 i 0.004 0.024 ± 0.0058 0.018 i 0.006 0.080 ±L 0.029 0.017 i 0.006 0.022 4 0.007

24 0.023 ± 0.006 0.020 ±i 0.008 0.022 i 0.004 0.026 i 0.006

aControl: K = 0.036 ±t 0.009 (50 determinations). Each value recorded represents the mean i 1 stand-ard deviation determined on five animals. Gelatin confers a continuous sustained clearance depressionon subsequently injected carbon.

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FUNCTION OF RETICULOENDOTHELIAL SYSTEM. IV

animals, a continuous and sustained period of de-pressed clearance resulted irrespective of the doseof carbon injected. Thus, gelatin addition elimi-nated the period of normal or accelerated clear-ance observed between the two periods of block-ade produced by an injection of 30 mg of carbonper 100 g. Further, gelatin addition to either asmall or a large carbon dose produced an effectequivalent to an injection of gelatin alone (Fig.4). Injection of the supernatant fluid of the origi-nal carbon preparation obtained after centrifuga-tion at 45,000 X g for 2 hr had no effect uponcarbon removal rate.

DISCUSSION

Phagocytic inhibition produced by colloid in-jection (RES blockade) is thought to be a singleperiod of RES paralysis due either to a depletionof blood opsonins (8, 16, 18) or to a cellularlimitation to further phagocytosis (1-3). In thepresent experiments, carbon injection produceda biphasic RES paralysis consisting of an earlytransient phase followed by a delayed sustainedphase. These experiments provide evidence thatRES blockade may be composed of at least twophases and each phase may involve a differentlimiting mechanism in phagocytosis. The phaseshave been designated as immediate or delayedparalysis to reflect their onset relative to the firstset of particles injected. Table 3 summarizes the

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essential differences between the two forms ofparalysis.Whereas two phases of RES paralysis were

demonstrated for carbon, the injection of a differ-ent type of particle, aggregated albumin, was fol-lowed by a single period of depressed clearance,which corresponded to the first phase of carboninduced inhibition. It would appear that thenature of the particles is an important factor inthe production of the second phase of paralysis.Since delayed paralysis is initiated after particleshave been cleared from the circulation, the pri-mary effect of the particles presumably is on thecell. The ability of the cell to catabolize the par-ticle may be an important factor because a me-tabolizable particle, such as aggregated albumin,would be expected to have different cellulareffects than inert particles such as carbon andthorotrast or toxic particles, such as silicon diox-ide (23), certain lipid emulsions (26, 27), or endo-toxin (7, 14). Indeed, a prolonged sustainedperiod of RES depression has been associatedwith injection of each of the latter type of par-ticles.

Injection of either carbon or aggregated al-bumin was followed by an immediate period ofRES paralysis. The duration of this effect was re-lated to the rate of particle removal, to theamount of particles injected, and to the presenceof the particles in the circulation. Therefore, inimmediate RES paralysis, there appears to be

o-o 10mg Carbon / lOOgm-^ ^30mg Carbon I lOOgm

5mg Gelatin Alone orK\'\\Iq 1 1mg Carbon + 5mg Gelatin I 100gm

TIME IN HOURS AFTER INJECTION

FIG. 4. Composite graph showing the effect of carbon, gelatin, and gelatin-carbon injection on the subsequentclearance of a standard carbon dose. The different preparations were injected only once, at time zero, and theeffect on the reticuloendothelial system determined in the time periods thereafter. Note that the larger carbon dose.without gelatin produces two distinct periods of clearance depression, whereas gelatin alone or gelatin with carbonproduces only a single sustained period ofdepressed clearance.

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TABLE 3. Summary of two types of particle-induced reticuloendothelial (RES) paralysis in rats

Characteristic Immediate RES paralysis Delayed RES paralysis

Onset Immediate, coincident with particle Delayed, develops sometime (hours)injection after particle injection

Particle types Metabolizable or inert Inert or toxicExamples of particles Carbon, aggregated albumin, gelatin Carbon, thorotrast, endotoxin, cer-

tain lipid emulsionsDuration Usually short (minutes to hours) but Prolonged (hours to days); duration

may be prolonged; duration de- not dependent upon dose or pres-pendent upon particle dose, rate of ence of circulating particlesremoval, and presence of particlesin circulation

Particle specificity Yes UnknownPossible mechanism Two-particle interaction in circula- Cellular derangement as (i) tempor-

tion as (i) adsorption onto particle ary failure to synthesize opsoninsurface, or (ii) competition for or replace cell constituents, or (ii)phagocytic sites or opsonin sustained failure requiring cell re-

placement for recovery.

some form of competition between the particlesin the circulation, whereas, in delayed RESparalysis, there appears to be a primary cellularderangement.The demonstration of two phases of RES

paralysis depended upon producing a sufficientlyshort period of immediate paralysis so that a re-covery period of normal or augmented clearanceexisted betwen the two phases of paralysis. Sucha short period of clearance inhibition might easilybe overlooked, since some particles of the firstinjection will be present in the circulation dur-ing this period. Adequate demonstration dependsupon an observed change in the clearance rateof a second particle injected during clearanceof the first. Thus, the clearance of DNS-labeledaggregated albumin was inhibited when injectedduring carbon clearance and, conversely, the clear-ance of carbon was inhibited when injected duringaggregated albumin removal. Furthermore, thesimultaneous determination of the clearance ratesof both particles showed that there is an inhibitionin the clearance rates of both particles (20). How-ever, not all particles would possess the necessaryproperties to compete with each other and a cer-tain degree of specificity in the phenomenonwould be expected; indeed, such specificity hasbeen described (13, 16, 25, 28).

Since the duration of immediate paralysis isdependent upon the numbers of particles injectedas well as their rates of clearance, it should bepossible to extend the period of immediateparalysis sufficiently so that an overlapping of thetwo periods of retarded clearance occurred. Gela-tin addition to the carbon suspension prior to in-jection of the suspension into animals slows therate of carbon removal. In addition, the presenceof gelatin, which is very slowly removed, can

itself retard the clearance of certain particles in-jected subsequently (13). Thus, the prolongedslow removal of carbon observed after gelatinaddition would be an example of the immediatetype of RES paralysis sustained by the slow re-moval of the first particle injected (gelatin). Be-cause gelatin can sustain a depression in carbonclearance rate, this fact alone, or the large carbondoses used, could account for the observations ofBiozzi et al. of only a single period of depressionafter carbon injection (2).

Recently, Jeunet and Good (9) suggested thatRES depression involves both cellular and hu-moral factors which could be dissociated in anisolated perfused liver system. Although theirconclusions are substantiated by the presentstudy, an important distinction between the twomodel systems should be pointed out. Whereasrepeated additions of aggregated albumin to anisolated perfused liver system produced an im-paired clearance at the cellular level, repeated in-jections of either aggregated albumin (19) orcarbon (4) in the intact animal results in fasterand faster rates of clearance. That the system canactively increase its appetite for inert particles oneach successive exposure in vivo indicates that,for all practical purposes, there is no limit to thecapacity of the system to engulf particles. This factrenders untenable the hypothesis that the causeof delayed RES paralysis could be due to anyform of cellular saturation (2), despite the his-toric acceptance of this theory. Therefore, adifferent form of cellular derangement must beinvolved. One possibility is a temporary failureof the cell to produce blood factors essential forphagocytosis. Pisano et al. (24) reported thatpuromycin pretreatment prevents recovery ofclearance rates depressed by injection of a test

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FUNCTION OF RETICULOENDOTHELIAL SYSTEM. IV

lipid emulsion and concluded that new synthesisof opsonic protein may be involved in recovery.

Alternatively, there could be a need for synthesisof new cell membrane or other cellular constitu-ents consumed in the process of phagocytosis.Finally with more extensive cellular derangement,there may need to be a replacement of cells toeffect recovery. Kelly and co-workers (11, 12)showed that ingestion of particles by cells resultsin a marked increase in incorporation of tritiatedthymidine into deoxyribonucleic acid of Kupffercells, and it has been reported that nitrogen mus-

tard, by blocking cell replication, can prolongRES blockade (1).The immediate RES paralysis that occurs when

two particles are in the circulation involves some

form of interaction between the particles. How-ever, it remains unclear how the presence of theparticles actually interferes with phagocytosis, al-though at least three possibilities suggest them-selves. (i) There is a direct interaction between theparticles, with one particle being adsorbed ontothe surface of the other, changing the surfaceproperties of either or both; (ii) there is a compe-

tition between the particles at the phagocyticsite on the cell (6); or (iii) there is a competitionfor, with resulting depletion of, available serum

factors essential to optimal phagocytosis (5, 15,18).

ACKNOWLEDGMMENTS

This investigation was supported under contract DADA17-68c-8125 with the United States Army Medical Research andDevelopment Command.

LITERATURE CITED

1. Benacerraf, B., B. N. Halpern, G. Biozzi, and S. A. Benos.1954. Quantitative study of the granulopectic activity ofthe reticuloendothelial system. III. The effect of cortisoneand nitrogen mustard on the regenerative capacity of theRES after saturation with carbon. Brit. J. Exp. Pathol.35:98-106.

2. Biozzi, G., B. Benacerraf, and B. Halpern. 1953. Quantitativestudy of the granulopectic activity of the reticuloendothelialsystem. II. A study of the kinetics of the granulopecticactivity of the RES in relation to the dose of carbon in-jected. Relationship between the weight of the organs andtheir activity. Brit. J. Exp. Pathol. 34:441-457.

3. Biozzi, G., C. Stiffel, B. Halpern, and D. Mouton. 1963.Lack of action of serum opsonins in phagocytosis of inertparticles by cells of reticuloendothelial system. Proc. Soc.Exp. Biol. Med. 112:1017-1020.

4. Dobson, E. L., L. S. Kelly, and-C. R. Finney. 1967. Kineticsof the phagocytosis of repeated injection of colloidalcarbon: Blockade, a latent period or stimulation? A ques-

tion of timing and dose, p. 63-73. In N. R. DiLuzio and R.Paoletti (ed.), The reticuloendothelial system and atherio-sclerosis. Plenum Press, New York.

5. Filkins, J. P., and J. J. Smith. 1965. Plasma factor influencingcarbon phagocytosis in the isolated perfused rat liver. Proc.Soc. Exp. Biol. Med. 119:1181-1184.

6. Fred, R. K., and M. L. Shore. 1967. Application of a mathe-matical model to the study of RES phagocytosis in mice,

p. 1-17. In N. R. DiLuzio and R. Paoletti (ed.), The reticulo-endothelial system and artheriosclerosis. Plenum Press,New York.

7. Heilman, D. 1965. The selective toxicity of endotoxin forphagocytic cells of the reticuloendothelial system. Int.Arch. Allergy 26:63-79.

8. Jenkins, C. R., and D. Rowley. 1961. The role of opsonins inthe clearance of living and inert particles by cells of thereticuloendothelial system. J. Exp. Med. 114:363-374.

9. Jeunet, F. S., and R. A. Good. 1969. Recticuloendothelialfunction in the isolated perfused liver. II. Phagocytosis ofheat aggregated bovine serum albumin. Demonstration oftwo components in the blockade of the reticuloendothelialsystem. Res. J. Reticuloendothel. Soc. 6:94-107.

10. Kampschmidt, R., H. Upchurch, and A. Park. 1966. Factorsin the suspending media which alter the carbon clearancerate. Res. J. Reticuloendothel. Soc. 3:214-221.

11. Kelly, L. S., B. A. Brown, and E. L. Dobson. 1962. Celldivision and phagocytic activity in liver reticulo-endothelialcells. Proc. Soc. Exp. Biol. Med. 110:555-559.

12. Kelly, L. S., E. L. Dobson, C. R. Finney, and J. D. Hirsch.1960. Proliferation of the reticuloendothelial system in theliver. Amer. J. Physiol. 198:1134-1138.

13. Koenig, M. G., R. M. Heyssel, M. S. Melly, and D. E. Rogers.1965. The dynamics of reticuloendothelial blockade. J.Exp. Med. 122:117-142.

14. Levy, E., and B. H. Ruebner. 1968. Hepatic changes inducedby a single dose of endotoxin in germfree mice. Amer. J.Pathol. 52:97-110.

15. Murray, I. M. 1963. Clearance rate in relation to agglutininsfor gelatin-stabilized colloid in the rat. Amer. J. Physiol.204:655-659.

16. Murray, I. M., 1963. The mechanism of blockade of reticulo-endothelial system. J. Exp. Med. 117:139-147.

17. Normann, S. J., and E. P. Benditt. 1965. Function of thereticuloendothelial system. I. A study of the phenomenonof carbon clearance inhibition. J. Exp. Med. 122:693-707.

18. Normann, S. J., and E. P. Benditt. 1965. Function of thereticuloendothelial system. II. Participation of a serumfactor in carbon clearance. J. Exp. Med. 122:709-719.

19. Normann, S. J., and E. P. Benditt. 1965. Reticuloendothelialblockade: The importance of the sequence and dose ofadministered particles. Res. J. Reticuloendothel. Soc. 2:345.

20. Normann, S. J., D. Lagunoff, and E. P. Benditt. 1968. Func-tion of the reticuloendothelial system. III. Simultaneousmeasurement of two particles during clearance inhibition.Lab. Invest. 19:353-357.

21. Snedecor, G. W. 1961. Statistical methods, p. 90-92. IowaState University Press, Ames, Iowa.

22. Parker, H. G., and C. R. Finney. 1968. Latent period in theinduction of reticuloendothelial blockade. Amer. J. Physiol.198:916-920.

23. Pearsal, N. N., and R. S. Weiser. 1968. The macrophage inallograft immunity. I. Effects of silica as a specific macro-

phage toxin. Res. J. Reticuloendothel. Soc. 5:107-120.24. Pisano, J. C., J. T. Patterson, and N. R. DiLuzio. 1968.

Reticuloendothelial blockade: Effect of puromycin on

opsonin-dependent recovery. Science 162:565-567.25. Schapiro, R. L., W. J. Maclntyre, and D. I. Schapiro. 1966.

The effect of homologous and heterologous carrier on theclearance of colloidal material by the reticuloendothelialsystem. J. Lab. Clin. Med. 68:286-299.

26. Stuart, A.E. 1962. Experimental necrosis of spleen. J. Pathol.Bacteriol. 84:193-200.

27. Stuart, A. E., C. Biozzi, C. Stiffel, B. N. Halpern, and D.Mouton. 1960. The stimulation and depression of reticulo-endothelial pathocytic function by simple lipids. Brit. J.Exp. Pathol. 41:599-604.

28. Wagner, H. N., Jr., and M. io. Studies of the reticuloendo-thelial system (RES). III. Blockade of the RES in man.J. Clin. Invest. 42:1525-1532.

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