Studies of the Effects of Administration of ACTHand Adrenal Corticosteroids onErythrophagocytosis

Robert M. Greendyke, … , Edwin M. Bradley, Scott N.Swisher

J Clin Invest. 1965;44(5):746-753. https://doi.org/10.1172/JCI105187.

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Journal of Clinical InvestigationVol. 44, No. 5, 1965

Studies of the Effects of Administration of ACTHand AdrenalCorticosteroids on Erythrophagocytosis *

ROBERTM. GREENDYKE,EDWIN M. BRADLEY, AND SCOTTN. SWISHERtWITH THE TECHNICAL ASSISTANCE OF NORMAC. TRABOLD

(From the Departments of Pathology and Medicine, University of Rochester School ofMedicine and Dentistry, Rochester, N. Y.)

Phagocytosis of effete erythrocytes or red cellfragments by reticuloendothelial cells appears toplay some role in normal red cell destruction, al-though the details of the process are poorly un-derstood. The marked increase in erythrophago-cytosis occurring in several types of hemolyticanemia, especially in acquired hemolytic diseaseand in some examples of transfusion reactions anderythroblastosis fetalis, indicates the possible im-portance of this process as a mechanism of redcell destruction. The rapid, often dramatic, sup-pression of erythrocyte destruction in patientswith acquired hemolytic disease frequently pro-duced by therapeutic adrenal corticosteroids sug-gests that these compounds may exert a specificeffect on phagocytosis of these abnormal erythro-cytes, apart from possible suppression of produc-tion of antibody-like proteins. The present studyextends preliminary observations (1) on the sup-pression of the in vitro erythrophagocytic capa-bilities of peripheral blood leukocytes obtainedfrom patients receiving ACTH or adrenal corti-costeroids.

Methods

The details of the basic experimental procedure em-ployed and a discussion of factors influencing in vitroerythrophagocytosis have been presented previously (2).The basic experimental procedure is as follows: Equalvolumes of a 3% suspension in 0.9%o saline of threetimes washed, fresh erythrocytes from a normal groupA donor and a suitable dilution of complement fixing

* Submitted for publication November 16, 1964; ac-cepted January 14, 1965.

This study was supported by research grant AM-00537-15, National Institute of Arthritis and MetabolicDiseases, and grant FR-44-03, Division of Research Fa-cilities and Resources, National Institutes of Health.

t Address requests for reprints to Dr. Scott N. Swisher,Hematology Unit, University of Rochester School ofMedicine and Dentistry, Rochester, N. Y. 14620.

anti-A antibody are combined. After incubation for 30minutes at 250 C, the cells are again washed three timesin saline and resuspended in 0.9% saline buffered to pH7.3 with one-fifth part isotonic phosphate buffer. Onevolume of a leukocyte suspension in fresh, autologousheparinized plasma containing 11,000 to 12,000 leuko-cytes and 20,000 to 30,000 erythrocytes per mm8 is added.The mixture is incubated for 1 hour at 370 C, gentlycentrifuged, and the cell sediment smeared and stainedwith Wright's stain. Five hundred neutrophils andmonocytes are examined, and the percentage exhibitingerythrophagocytosis is determined.

Preliminary studies of the effect of adrenal cortico-steroids added to leukocyte suspensions in vitro were car-ried out by these methods. Aqueous solutions of solublecorticosteroids were added to human leukocyte suspen-sions in fresh heparinized autologous plasma, and themixture was incubated for varying periods of time at250 C. At intervals, samples of this mixture were addedto suspensions of human group A erythrocytes previouslyopsonized. The experiments were then completed asabove. pH values in the supernatant fluid of experimentaland control tubes were determined. Complement levelswere assayed by adding dilutions of this supernatantfluid to sensitized sheep red cells. The resulting hemoly-sis was read visually. In some studies, the leukocyteswere washed and resuspended in fresh autologous plasmabefore being added to sensitized erythrocytes. Controlstudies were carried out to rule out effects of the com-pounds added on the opsonification portion of the over-all procedure.

Experiments were carried out to ascertain the dailyand diurnal variation of erythrophagocytic capacity ofthe leukocytes of a number of donors. These studieswere carried out by obtaining serial blood samples fromhuman experimental subjects at intervals; combiningleukocyte suspensions in fresh heparinized autologousplasma from these samples with group A1 erythrocytespreviously sensitized with anti-A, washed, and suspendedin buffered saline; and completing the experiments asabove. In certain studies, the leukocyte suspensionswere prepared in plasma obtained from compatible do-nors. Because of the critical dependence of the amountof erythrophagocytosis on the degree of opsonificationof the erythrocytes in these studies, samples from a singlelot of sensitized red cells were used throughout indi-vidual experiments. Rigidly quantitative technique was

746

ADRENALCORTICOSTEROIDSANDERYTHROPHAGOCYTOSIS

employed throughout in preparation and measurement ofall cell samples, with adjustment of the leukocyte countof the leukocyte suspensions to comparable values(11,000 to 12,000 per mm8) before use.

Experiments involving infusion of ACTH into hu-man volunteers included a sham intravenous infusionof 700 to 800 ml of 5% detrose in water over a 6- to 8-hour period on one day and the intravenous infusion of

60

50

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0

300

I0

LI>. 20

LU

20 U of ACTHin a similar volume of dextrose and wa-

ter the following day. Small samples of venous bloodfor determination of leukocyte erythrophagocytic abilitywere obtained at intervals throughout these infusions,and approximately 3 hours after the start of each in-fusion a sample of arterial blood was obtained for de-termination of plasma cortisol levels.

A similar protocol was followed for experiments on

TIME (HRS.)FIG. 1. EFFECT ON ERYTHROPHAGOCYTOSISOF INCUBATING LEUKOCYTES IN

FRESH HEPARINIZED AUTOLOGOUSPLASMA WITH EQUAL AMOUNTSOF ISOTONICSALINE SOLUTIONS, PH 7.4, OF CORTISOL (0.5 AND 2 MGPER ML), PREDNISOLONE(0.05 AND 0.2 MGPER ML), OR ISOTONIC SALINE ALONEFOR VARYING PERIODS OFTIME BEFORE COMBINATION WITH OPSONIZED ERYTHROCYTESFOR A STANDARDPERIOD OF PHAGOCYTOSIS. The decline in erythrophagocytic ability of leuko-cytes aged in plasma plus saline is due, at least in part, to accumulation of in-hibitory substances in the suspending plasma (4).

747

R. M. GREENDYKE,E. M. BRADLEY, AND S. N. SWISHER

the effect of intravenously administered cortisol. Bymeans of a constant infusion pump, cortisol (hydrocor-tisone, alcohol U.S.P.) was infused into an arm vein ata rate of 14 ug per minute, or 160 Ag per minute, forlow and high dose levels, respectively. The duration ofthe infusions was 4 to 6 hours. Aldosterone alcohol wasinfused in similar fashion at the rate of 0.012 ltg per min-ute for 4 hours in a single experiment. In the studies onthe effects of prednisone, the drug was administered

-orally in a single dose of 80 mg, and the above generalexperimental outline was then followed.

Quantitative estimation of the concentrations of plasmacortisol were carried out by the method of Plager, Cush-man, and Chase (3). Blood was collected in heparinizedsyringes. The plasma was separated within 30 minutesand stored frozen until analyzed. Prednisone levels weredetermined in a similar fashion except that the determina-tion was performed on the eluate of the prednisone areaof the paper chromatogram.

Results

A series of experiments was carried out to de-termine the effect on erythrophagocytosis of in-cubating in vitro normal leukocytes suspended infresh autologous heparinized plasma with varyingconcentrations of cortisone acetate, cortisol, de-soxycorticosterone acetate (DOCA), and predni-solone for varying periods of time before com-bination with opsonized erythrocytes. Such ex-periments showed no inhibition by DOCA inconcentrations up to 0.5 mg per ml. Inhibition ofleukocyte erythrophagocytic ability by the three

TABLE I

Erythrophagocytosis*

Donor no. Experiment 1 Experiment 2 Experiment 3

1 64 68 642 82 84 813 68 72 684 67 705 25 29 316 47 41 417 43 43 468 53 57 589 22 20 25

10 44 42 4611 56 57 5912 53 55 50

Mean and SD 52.0 i 0.7 52.9 i 0.7 53.3 + 0.7

* Erythrophagocytic ability of leukocytes from 12 fastinghealthy adult donors of blood group A1 bled simultaneouslyon three different occasions 5 days apart. The differencesbetween the mean values of the three sets of experimentsare not statistically significant. The differences fromdonor to donor are highly significant (p < 0.001). TheSD of a single observation is estimated to be 2.4%.

glucocorticoids was not demonstrated until grosslyunphysiological quantities of the test compound (ofthe order of 0.2 to 2.0 mg per ml) were added tothe incubation medium. These experiments donot lend themselves to tabulation or statisticaltreatment because of the variability from one toanother of degree of erythrocyte opsonification andphagocytic activity of donor leukocytes, resultingin differences in base-line values and rates of in-hibition of erythrophagocytosis. The results of arepresentative experiment demonstrating inhibi-tion of erythrophagocytosis by in vitro incubationof leukocytes suspended in plasma with addedprednisolone or cortisol are shown in Figure 1.With the assay employed, no inhibition of comple-ment activity by the test compound as comparedwith normal saline controls was found in theseexperiments. A modification of these studies wasalso carried out; after preincubation of the leuko-cytes in plasma plus steroid (or normal saline asa control) for intervals of up to 12 hours, the leu-kocytes were washed twice and resuspended inautologous heparinized plasma before addition ofopsonized erythrocytes. Under these conditions,leukocytes preincubated with cortisol (2 mg perml) or prednisolone (0.2 mg per ml) exhibitedthe same phagocytic ability as did the controls inwhich saline had been added to the suspendingplasma instead of to the test compound.

Before studies on the effects of corticosteroidsadministered in vivo were begun, a series of pre-liminary investigations was carried out to deter-mine the variation with time in erythrophagocyticability of leukocytes from various donors. Four-teen experiments were conducted in which normalvolunteers (nine subjects) or patients with mod-erately severe chronic adrenal cortical insufficiency(two subjects) were bled at 1- to 4-hour intervalsbetween 9:00 a.m. and midnight, while at rest,either with or without the administration of an

intravenous infusion of glucose in water duringthis time. No diurnal variation was demon-strated in leukocyte erythrophagocytic ability inany subject when fresh leukocytes were employed.

Another study was carried out to determinethe variability of leukocyte erythrophagocyticability among a group of healthy adults both fromthe viewpoint of donor to donor variability at a

given time of day and variability in individual

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ADRENALCORTICOSTEROIDSAND ERYTHROPHAGOCYTOSIS

donors studied at the same time on different days.Twelve healthy adult donors, seven females andfive males, aged 20 to 65 years, all of erythrocytegroup A, and all with normal total and differentialleukocyte counts, were simultaneously bled afteran overnight fast on each of three occasions sepa-rated by 5 days each. Leukocyte suspensionswere prepared from each sample, adjusted to auniform concentration, and combined with samplesof opsonized A, erythrocytes from a single donor.The variability in erythrophagocytic ability fromdonor to donor and the uniformity of this abilityin leukocytes obtained from single donors on dif-ferent days are demonstrated by the results shownin Table I. The complement titers of all donorson all three occasions were the same.

To ascertain whether the differences observedin the erythrophagocytic ability of leukocytesfrom various donors were due to serum factorsor to primary differences among the leukocytes,leukocyte suspensions were prepared as abovefrom 10 of the 12 healthy adult donors previouslymentioned. The leukocytes were washed twicein saline, and samples of each were resuspended ina) fresh heparinized autologous plasma, b) freshheparinized plasma from Donor 2, whose leuko-

TABLE II

Erythrophagocytosis*

Leukocytes Leukocytes Leukocytesin autol- in plasma in plasma

Donor no. ogous plasma from Donor 2 from Donor S

1 68 74 422 81 84 593 69 79 634 70 74 525 30 57 337 46 62 338 60 69 529 29 67 30

11 60 68 4312 49 55 30

Mean and SD 56.2 i 2.1 68.9 4t 2.1 43.7 i 2.1

* Erythrophagocytic ability of leukocytes from ten fast-ing healthy adult donors of blood group Al bled simultane-ously. The leukocytes were washed and resuspended inautologous plasma, and in plasma from donors showinghigh (Donor 2) and low (Donor 5) erythrophagocyticability of their leukocytes when suspended in their respec-tive autologous plasmas. Samples of opsonized erythro-cytes from a single donor were employed in all cases.

The results in the second column are significantly higher(t = 4.29 with 18 degree of freedom, p < 0.001) and in thethird column are significantly lower (t = 4.23 with 18degrees of freedom, p < 0.001) than in the first column.The SDof a single observation is estimated at 6.6%.

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0z 10F

Lu-2 0 2 4 6 8 10 12

TIME FROM START OF INFUSION (HRS.)

FIG. 2. EFFECT ON ERYTHROPHAGOCYTOSISOF INTRA-VENOUS INFUSION OF SALINE OR 20 U OF ACTH OVERA 6-HOUR PERIOD TO NORMALLEUKOCYTEDONOR.

cyte suspensions in autologous plasma had previ-ously shown very active erythrophagocytic ability,and c) fresh heparinized plasma from Donor 5,whose leukocyte suspensions in autologous plasmahad shown very low erythrophagocytic ability.Samples of opsonized erythrocytes from a singledonor were added and the experiments completed.The results of this study are shown in Table II;it will be seen that enhancement of erythrophago-cytic ability, averaging 38%o, was obtained by in-cubating various donors' leukocytes in plasmafrom Donor 2, and depression of an average of23%o was effected by plasma from Donor 5.Again, no differences in complement activity inthe plasma samples from any of the donors weredemonstrated by the method utilized.

In a series of experiments on the effect of invivo administration of ACTH, a total of eightstudies was carried out on five normal subjects,and two studies were performed using two pa-tients with well documented chronic adrenal cor-tical insufficiency. The typical alteration in eryth-rophagocytic ability of leukocytes from a normalsubject during intravenous ACTH infusion andthe lack of this effect during infusion of glucosein water are shown in Figure 2. The plasma cor-tisol values for this subject 3 hours after the startof the ACTH infusion were 34.5 ug per 100 ml.Normal plasma cortisol levels without adrenalstimulation by this method are less than 10 pgper 100 ml. Similar results were obtained in each

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R. M. GREENDYKE,E. M. BRADLEY, AND S. N. SWISHER

TABLE III

Effect on erythrophagocytic ability of ACTH infusion infive normal subjects on eight occasions*

Maximal de-crease in

erythrophago-cytosis rela- Plasmative to ini- cortisol

Patient tial value concentration

% yjg/100 ml

W. H. -55N. H. -50 34.5N. H.t -47 79.0W. S. -28 53.2W. S.t -26 98.8H. B. -50 17.2H. K. -39 47.9H. K. -48 52.3

* The maximal effect was noted approximately 3 hoursafter the infusion was begun. Plasma cortisol levels weredetermined at this time.

t Patients N. H. and W. S. were restudied after 4 and 2weeks, respectively, of intensive estrogen therapy. Therise in plasma cortisol levels is thought to be presentchiefly in the protein-bound fraction and not available toinfluence leukocyte function as much as would a compar-able rise in unbound cortisol (5).

of the seven other studies of this type that wereconducted. Maximal depression of erythrophago-cytic ability was present within 3 hours after thestart of the ACTH infusion, the drop averaging43% of the initial values. The percentage fall inerythrophagocytosis between the start of theACTH infusion and the time of maximal depres-sion plus the concentrations of plasma cortisol 3hours after the start of the infusion in these stud-

3

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FIG. 3. EFFECT ON ERYTHROPHAGOCYTOSISOF INTRA-

VENOUSINFUSION OF 55 MGOF CORTISOL OVER A 6-HOURPERIOD TO A NORMALLEUKOCYTEDONOR.

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0(9orct 20

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FIG. 4. EFFECT ON ERYTHROPHAGOCYTOSISOF INTRA-VENOUSINFUSION OF 65 MGOF CORTISOL OVER A 6i HOURPERIOD TO A PATIENT WITH CHRONIC ADRENAL CORTICALINSUFFICIENCY.

ies are listed in Table III. No significant vari-ation in leukocyte type or maturity was demon-strated in the course of these studies to explain theresults observed.

Similar studies in two patients with chronicadrenal cortical insufficiency normally maintainedon 17.5 and 25 mg of cortisone per day, respec-tively, disclosed that ACTH infusion exerted noeffect on the erythrophagocytic ability of leukocytesobtained from these subjects, the amount of eryth-rophagocytosis remaining unchanged throughoutthe studies. Both patients had received no ex-ogenous adrenal cortical hormone or analog for 24hours before the test was begun. The plasma cor-

tisol levels of the patient in the experiment shownwere 8.2 Mg per 100 ml 3 hours after the ACTHinfusion was begun. The erythrophagocytic abilityof the leukocytes of each of these two patients waslater shown to be depressed normally by intra-venously administered cortisol or orally adminis-tered prednisone.

Cortisol was administered in a dose of 55 mgintravenously during a period of 6 hours to a

normal subject, with the effects on leukocyteerythrophagocytosis shown in Figure 3. Theplasma cortisol level 3 hours after the start of theinfusion was 36.4 Mg per 100 ml. Similarly, a pa-tient with chronic adrenal cortical insufficiencynormally maintained on 17.5 mg of cortisone daily,

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ADRENALCORTICOSTEROIDSANDERYTHROPHAGOCYTOSIS

who had received no medication for 24 hours, wasgiven 65 mg of cortisol intravenously over a (4-hour period, with the results shown in Figure 4.Three hours after the start of the infusion theplasma cortisol level was 65.6 ug per 100 ml. Inboth cases, sham infusions on the previous daycaused no alteration in erythrophagocytosis. Twosimilar studies on normal subjects and one studyon a patient with chronic adrenal cortical insuffi-ciency in which physiological amounts of cortisol,16 e1g per minute, were infused for 6 hours showedno effect on erythrophagocytosis and did not raisethe plasma cortisol levels above normal.

Figures 5 and 6 show the effects on the erythro-phagocytic ability of peripheral blood leukocytesof oral administration of a single 80-mg dose ofprednisone to a normal volunteer (Figure 5) andto a patient with chronic adrenal cortical insuffi-ciency (Figure 6). The latter patient was nor-mally maintained on 25 mg of cortisone daily andhad received no medication for 24 hours. Theplasma prednisone levels in the two studies 3 hoursafter administration of the drug were 44.6 and61.0 jig per 100 ml, respectively.

90j.

A,_ TV

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0

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30

i 20

I0

-2 0 2 4 6 8 l0 12

TIME AFTER ADMINISTRATION OF DRUG(HRS)

FIG. 5. EFFECT ON ERYTHROPHAGOCYTOSISOF ORAL AD-

MINISTRATION OF A SINGLE 80-MG DOSE OFPREDNISONETO

A NORMALLEUKOCYTEDONOR.

zuJ0 70

LLJ

a 60

InC,)o 50

0(5 40

0° 30

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-2 0 2 4 6 8 l0 12TIME AFTER ADMINISTRATION OF DRUG (HRS.)

FIG. 6. EFFECT ON ERYTHROPHAGOCYTOSISOF ORAL AD-MINISTRATION OF A SINGLE 80-MG DOSE OF PREDNISONETOA PATIENT WITH CHRONIC ADRENAL CORTICAL INSUFFI-CIENCY.

Other experiments were carried out in whichleukocytes obtained before the start of an ACTHor "high concentration" cortisol infusion and leu-kocytes obtained 4 hours after the start of theinfusion were washed and samples of each resus-pended in both the "0-hour" and "4-hour" plasma.The decreased erythrophagocytic ability found in4-hour leukocytes suspended in 4-hour plasmawas returned to the original higher value by sus-pension in 0-hour plasma; conversely, 0-hourleukocytes suspended in 4-hour plasma showedinhibition of erythrophagocytosis comparable tothat seen in 4-hour leukocytes suspended in 4-hourplasma. Studies of this type during three ACTHinfusion tests on normal subjects and one highconcentration cortisol infusion in a patient withchronic adrenal cortical insufficiency yielded com-parable results.

A single patient with chronic adrenal corticalinsufficiency, previously shown to be responsive inerythrophagocytic studies to large amounts of in-travenously administered cortisol, was given anintravenous infusion of 2.9 mg of aldosterone

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751

9c

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R. M. GREENDYKE,E. M. BRADLEY, AND S. N. SWISHER

over a 4-hour period. Serial tests of erythro-phagocytic ability of his peripheral blood leuko-cytes during the infusion and for 3 hours after itstermination showed no change.

Discussion

Considerable attention has been paid by in-vestigators to elucidation of the effects of thera-peutic ACTHand adrenal corticosteroids on theprocess of phagocytosis. Early observations on

the deleterious effects of glucocorticoid adminis-tration in many infectious diseases led to extensivestudies of effects of steroid compounds on theclearance of various intravenously injected ma-

terials by the reticuloendothelial system of theintact animal. Inconclusive or contradictory re-

sults were obtained in certain of the studies, some

of which showed increased and others decreasedphagocytic activity by the fixed reticuloendothelialelements (6-11) under the influence of gluco-corticoids or ACTH. Similar inconsistent effectsof glucocorticoids or ACTH were noted by in-vestigators working with phagocytosis of bacteriaby exudate macrophages and blood leukocytes(12-18). All investigators appear to agree on

the lack of effect on phagocytosis of corticosteroidcompounds lacking glucocorticoid activity. Aspointed out by the work of Crabbe (14-16), Rei-chard, Edelmann, and Gordon (8, 9) and Snell(11), most of the discrepancies seem to be ac-

counted for by differences in phagocytic activitytoward various types of test materials and by thegeneral pharmacological principle that many com-

pounds that stimulate in low concentrations are

inhibitory at higher levels. It should be empha-sized that almost none of the previous work inthis area has included actual measurement of bloodcorticosteroid concentrations at the time that phago-cytic capabilities were being assessed; the stud-ies imply the presence of elevated corticosteroidconcentrations in the experimental situations butgive no quantitative supporting data. Further,as previously emphasized, little of the previouswork on the subject of phagocytosis has con-

cerned itself with various effects as variables withtime, a situation leading to a number of discrep-ancies in the older literature (4).

The mechanisms by which glucocorticoids bringabout their multiple physiological effects are poorly

understood, but seem to revolve about "permis-sive" effects on the permeability of cellular andintracellular membranes (5). In light of the pre-dominantly glycolytic metabolism of peripheralblood leukocytes, it is of interest to note the workof Martin, Chaudhuri, Green, and McKinney(19), confirmed by Rauch, Loomis, Johnson, andFavour (20), demonstrating marked inhibitionof glycolysis effected by in vitro incubation of in-tact human peripheral blood leukocytes with mi-crogram per milliliter concentrations of certainglucocorticoids, and the lack of such effects whenthe cells were disrupted (20). Previous studiesfrom this laboratory have noted the inhibition oferythrophagocytosis by in vitro incubation of leu-kocytes with other inhibitors of glycolysis (2),suggesting that the pharmacologic effect of gluco-corticoids in suppressing phagocytosis in vivo atleast by peripheral blood cells may be accomplishedthrough an ultimate effect on the energy yieldingmetabolism of the leukocyte. That this is not asimple phenomenon, however, is indicated by thegross quantitative differences in the amount ofglucocorticoid necessary to produce inhibition oferythrophagocytosis when incubated in vitro withthe leukocyte suspension as opposed to the amountsnecessary to produce inhibition when administereddirectly to the leukocyte donor.

A question may be raised over whether the glu-cocorticoid inhibition of erythrophagocytosis ob-served in the present studies could be explained bydifferences in the maturity, and hence phagocyticavidity, of the neutrophils. It would appear thatthe experiments in which plasma containing highlevels of administered glucocorticoids inhibitedcells from blood containing normal levels of glu-cocorticoids, and vice versa, argue against this asa major explanation for the effects demonstrated.

The experimental results obtained in this workindicate that the phenomenon of erythrophago-cytosis is very responsive to the presence of glu-cocorticoid compounds in the circulating bloodand suggest the possibility that simple modifica-tions of these experiments might serve as excel-lent clinical tests of adrenal cortical responsivenessto ACTH.

SummaryExperiments were conducted to assess the ef-

fects of ACTHand certain adrenal corticosteroids

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ADRENALCORTICOSTEROIDSANDERYTHROPHAGOCYTOSIS

on the in vitro phagocytic capability of humanperipheral blood leukocytes for opsonized eryth-rocytes. In vitro addition of glucocorticoids re-quired grossly unphysiological amounts of thesesubstances to cause inhibition. In contrast, in-travenously administered cortisol and orally ad-ministered prednisone caused sequential decreasein the erythrophagocytic capacity of leukocytesfrom both normal subjects and patients withchronic adrenal cortical insufficiency. Intrave-nously administered ACTHshowed a similar ef-fect in normal patients but yielded no effect inpatients with Addison's disease. It is suggestedthat the techniques employed may lend themselvesto clinically useful methods for assessing certainaspects of adrenal glucocorticoid metabolism.

Acknowledgment

The authors gratefully acknowledge the assistance ofDr. A. Dutton in performing the statistical analysis inthis study.

References

1. Packer, J. T., R. M. Greendyke, and S. N. Swisher.Inhibition of erythrophagocytosis in vitro by cor-ticosteroids. Trans. Ass. Amer. Phycns 1960,73, 93.

2. Greendyke, R. M., R. E. Brierty, and S. N. Swisher.In vitro studies on erythrophagocytosis. Blood1963, 22, 295.

3. Plager, J. E., P. Cushman, Jr., and A. E. Chase.The plasma cortisol response to ACTH in "idio-pathic hirsutism." J. clin. Invest. '1961, 40, 1315.

4. Greendyke, R. M., R. E. Brierty, and S. N. Swisher.In vitro studies on erythrophagocytosis. II. Ef-fects of incubating leukocytes with selected cellmetabolites. J. Lab. clin. Med. 1964, 63, 1016.

5. Bush, I. E. Chemical and biological factors in theactivity of adrenocortical steroids. Pharmacol.Rev. 1962, 14, 317.

6. Gordon, A. S., and G. F. Katsh. The relation of theadrenal cortex to the structure and phagocyticactivity of the macrophagic system. Ann. N. Y.Acad. Sci. 1949, 52, 1.

7. Heller, J. H. Cortisone and phagocytosis. Endo-crinology 1955, 56, 80.

8. Reichard, S. M., A. Edelmann, and A. S. Gordon.Adrenal and hypophyseal influences upon the up-take of radioactive gold (Aul') by the reticulo-endothelial system. Endocrinology 1956, 59, 55.

9. Reichard, S. M., A. Edelmann, and A. S. Gordon.Endocrine influences upon the uptake of colloidalthorium by the reticuloendothelial system. J. Lab.clin. Med. 1956, 48, 431.

10. Nicol, T., and D. L. J. Bilbey. Substances de-pressing the phagocytic activity of the reticulo-endothelial system. Nature (Lond.) 1958, 182,606.

11. Snell, J. F. The reticuloendothelial system. I.Chemical methods of stimulation of the reticulo-endothelial system. Ann. N. Y. Acad. Sci. 1960,88, 56.

12. Crepea, S. B., G. E. Magnin, and C. J. Seastone.Effect of ACTH and cortisone on phagocytosis.Proc. Soc. exp. Biol. (N. Y.) 1951, 77, 704.

13. Moeschlin, S., W. Zurukzoglu, and J. Crabbe. Un-tersuchungen uber den Einfluss von Cortison undACTH auf die Phagozytose der Leukozyten undMakrophagen. Acta haemat. (Basel) 1953, 9, 277.

14. Crabbe, J. The effects of cortisone on the phagocyticactivity of rabbit macrophages to staphylococci.Acta endocr. (Kbh.) 1955, 18, 251.

15. Crabbe, J. The effects of ACTHon the phagocyticactivity of rabbit macrophages to staphylococci.Acta endocr. (Kbh.) 1955, 18, 256.

16. Crabbe, J. Enhancing action of small doses of cor-tisone on macrophage phagocytosis of staphylo-cocci in rabbits. Acta endocr. (Kbh.) 1956, 21,41.

17. Ludany, G., J. Rigo, J. Sos, and G. Vajda. Predni-solon-Na-succinat und die Phagozytose der Leu-kozyten. Experientia (Basel) 1959, 15, 463.

18. Hirsch, J. G., and A. B. Church. Adrenal steroidsand infection: the effect of cortisone administrationon polymorphonuclear leukocytic function and onserum opsonins and bactericidins. J. clin. Invest.1961, 40, 794.

19. Martin, S. P., S. N. Chaudhuri, R. Green, and G. R.McKinney. The effect of adrenal steroids onaerobic lactic acid formation in human leukocytes.J. clin. Invest. 1954, 33, 358.

20. Rauch, H. C., M. E. Loomis, M. E. Johnson, andC. B. Favour. In vitro suppression of poly-morphonuclear leukocyte and lymphocyte glycoly-sis by cortisol. Endocrinology 1961, 68, 375.

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