[CANCER RESEARCH 44, 3936-3941, September 1984]

In Vitro Effects of 4-Hydroperoxycyclophosphamide on the Morphology

and Function of Human Peripheral Blood Mononuclear PhagocyticCells (Macrophages)1

Mark Jason, Brian S. Andrews,2 Michael Colvin, and George J. Friou

Department ol Medicine, University of California, Irvine, California [M. J., B. S. A., G. J. F.], and Johns Hopkins University School of Medicine, Baltimore, Maryland[M. C.]

ABSTRACT

Human peripheral blood mononuclear cells were incubated for1 hr in 4-hydroperoxycyclophosphamide (0.5 to 10.0 ng/m\), andthe adherent, esterase-positive cells (macrophages) were stud

ied. At 2 hr, a reduction was noted in both latex particle ingestionand Fc., receptor binding and phagocytosis. At 24 hr, spreadingand pinocytosis were reduced, and cytoplasmic vacuoles developed. This vacuolization represented dilatation of the roughendoplasmic reticulum. These morphological and functionalchanges occurred with 4-hydroperoxycyclophosphamide con

centrations which did not reduce viability or produce detectableDNA alkylation. This effect on macrophages may offer a mechanism whereby low-dosage cyclophosphamide could modify the

immune response.

INTRODUCTION

CY3 is a potent alkylating agent used in cancer chemotherapy

(12) and as an immunosuppressive agent in the management ofa variety of inflammatory rheumatic diseases (1, 2, 7, 11, 28).CY requires biochemical conversion by hepatic microsomal P-450 enzymes to the metabolically active product 4-HCY, which

then undergoes further transformation to active alkylating products (12). While in vivo studies can be undertaken with CY, invitro studies have been limited by the lack of availability of anactive agent. The synthetic material 4-HPCY, which is spontaneously hydrolyzed in solution to 4-HCY, has been used in

several recent in vitro studies (6, 15, 19). Kaufmann ef a/. (15)and Diamanstein ef al. (6), using the DTH and antibody responsesin mice to sheep erythrocytes, documented a differential sensitivity of T-lymphocyte subpopulations to 4-HPCY. They notedthat, at lower doses of 4-HPCY, suppressor T-lymphocytesinvolved in DTH (15) and helper T-lymphocytes of the humoral

immune response were eliminated (6, 15), with minimal effectson helper T-lymphocytes of DTH and suppressor T-lymphocytesof the humoral immune response. Ozer ef a/. (19), using 4-HPCY-

treated human lymphocytes, noted a similar differential sensitivityof lymphocyte subpopulations in a polyclonal B-cell differentiationassay and in the generation of Con A-induced suppressor cells

1Supported by the Southern California Chapter of the Arthritis Foundation and

an Arthritis Foundation Clinical Research Center grant.2To whom requests for reprints should be addressed, at Division of Rheuma

tology and Clinical Immunology, Department of Medicine, Medical Sciences I,University of California, Irvine, CA 92717.

3The abbreviations used are: 4-HPCY, 4-hydroperoxycyclophosphamide; CY,cyclophosphamide; 4-HCY, 4-hydroxycyclophosphamide; DTH, delayed-type hy-persensitivity; Con A, concanavalin A; PBM, peripheral blood mononuclear cells;HRP, horseradish peroxidase; EA,, IgG-sensitized sheep erythrocytes; RER, roughendoplasmic reticulum.

Received January 9, 1984; accepted May 31, 1984.

for effector B-lymphocyte function. In low 4-HPCY concentrations, where measurable DNA cross-linking and decreased blas-togenesis could not be detected, 4-HPCY selectively inhibitedthe differentiation of presuppressor (OKT4+, OKT8~) to suppres

sor cells. At higher 4-HPCY concentrations, T-cell inducers be

came sensitive, whereas differentiated suppressor effector function was resistant, except at the highest concentrations. Furthermore, B-cell subpopulations also appeared to be differentiallysusceptible to 4-HPCY concentrations (19).

In human in vivo studies, the effects of CY on immunologicalfunction have been conflicting. In part, this relates to: (a) theselection of patients (disease treated, duration of treatment); (b)differing doses and modes of administration of CY (ultimately,the dose and the presumed CY metabolite levels in serum isbased on the resultant WBC); and (c) the frequent use ofcorticosteroids in combination, although it is claimed that corti-costeroids alone do not influence lymphocyte function whenstudied under certain situations (10, 14). Taube ef al. (23) notedthat patients with minimal-change nephropathy treated with CY

were unable to generate Con A suppressor cell function and thatthis impairment correlated with a good therapeutic response. Ina recent study using chronic low-dose CY in patients with

systemic vasculitis, Cupps ef al. (5) demonstrated a selectivesuppression of B-cell function determined by PWM-induced im-

munoglobulin secretion.In all of the above studies, the role of the macrophages has

not been addressed. Macrophages play a pivotal role in theinduction, regulation, and expression of the immune response(18, 20, 26). They ingest, process, and present antigens tolymphocytes in association with la antigens; this ultimately leadsto lymphocyte activation. Furthermore, macrophages serve aregulatory function in the helper-suppressor lymphocyte inter

action, are involved in expression of the immune response, e.g.,by release of soluble mediators such as interleukin I, contributeto host defense mechanisms, and may produce tissue destruction following release of lysosomal enzymes (17). Thus, an effectof CY on this cell population(s) may significantly influence theinterpretation of the data generated from in vivo and in vitrostudies using CY.

MATERIALS AND METHODS

Cell Preparation. PBM were obtained from 7 healthy normal volunteers and studied on 3 occasions. PBM were isolated by density gradientcentrifugation using heparinized venous blood (20 units of heparin perml) on cushions of sodium diatrizoate-Ficoll (LSM; Bionetics Laboratory

Products, Kensington, MD) (16). The mononuclear cells were then removed and washed 3 times in RPM11640 containing L-glutamine (2 mw),

Fungizone (2.5 M9/ml), and gentamicin (10 M9/ml) (RPM11640 medium).Cells (107/ml) were then incubated in triplicate with 4-HPCY in RPMI

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Macrophages and CY

1640 medium at concentrations ranging from 0.1 to 100 ^g/m\ and inmedium alone for 1 hr at 37°in 12- x 75-mm sterile polypropylene tubes

(Becton Dickinson, Oxnard, CA). After the incubation period, the cellswere pelleted and washed thoroughly 3 times at 4°in RPM11640 medium

containing 10% fetal calf serum; this medium was used for the remainderof the experiments. Care was taken to minimize macrophage adherenceto the tube by frequent agitation during the incubation and washing thetube after the pellet had been removed. PBM preparations containing 2x 106 macrophages in a volume of 250 tf, as determined by esterase

staining, were then added to a sterile plastic coverslip in the form of abubble on the center of the coverslip. The coverslip was then incubatedin a humidified incubator for 2 hr at 37°to allow macrophage adherence.

Cell viability was determined in the initial cell preparation and at 2 and24 hr after adherence on the adherent cell and nonadherent cell populations. The nonadherent cells were maintained in medium at 37° in

sterile polypropylene tubes after the initial 2-hr incubation, and viability

was assessed at 24 hr. Viability was determined using ethidium bromide(8) and fluorescein diacetate (9). Cell suspensions were incubated withthe fluorescent probes (2 and 5 ^g/ml, respectively) for 10 min at roomtemperature, and the number of viable cells was determined by fluorescent microscopy.

4-HPCY. 4-HPCY was synthesized by Michael Colvin at the JohnsHopkins Oncology Center, Baltimore, MD. 4-HPCY is relatively unstable,is maintained as a desiccated powder at -20°, and is prepared in a

stock solution in RPMI 1640 medium immediately prior to incubationwith the mononuclear cells. 4-HPCY spontaneously hydrolyzes inaqueous solution to 4-HCY, the compound formed in the liver following

microsomal activation of CY.Light Microscopy. A cytocentrifuge (Cytospin, Shandon Southern,

Sewickley, PA) preparation of PBM was obtained and stained withWright's (Sigma Chemical Co., St. Louis, MO) and esterase stains (3).

Stains were performed on the initial PBM preparation and on adherentand nonadherent cells at 2 and 24 hr.

Electron Microscopy. Macrophages which had adhered to plasticcoverslips were washed in normal saline and fixed with 2.5% glutaral-dehyde in 0.05 M phosphate-buffered saline, pH 7.4, for 60 min at room

temperature. After dehydration, propylene oxide was added to dissolvethe plastic coverslip (25). After detachment, the cells were harvested,transferred to glass tubes, concentrated into pellets by centrifugation,and embedded in Poly-Bed (Polysciences, Warrington, PA). Thin sectionswere cut on a Riehen OM-U2 microtome, stained with uranyl acetate

and lead citrate, and examined with a Zeiss 9S2 electron microscope.Macrophage Adherence. The number of esterase-positive macro

phages was determined prior to addition of the isolated PBM to theplastic coverslip. After incubation on the coverslip for 2 hr at 37°, the

supernatant was carefully collected, and the coverslip was washed 3times with medium. All washes were saved, and the total number ofPBM was determined in the nonadherent cell population. An esterasestain was then performed on the nonadherent PBM to determine thetotal number of nonadherent macrophages. The percentage of adherentmacrophages was then determined (24) by the formula

No. of macrophages in initial sample- no. of macrophages in nonadherent cells

No. of macrophages in initial samplex 100

Adherence rates for control, normal, untreated macrophages rangedfrom 80 to 90%.

Spreading of Adherent Macrophages. Spreading of the adherentmacrophage population was determined after 24 hr of incubation. Afterthe initial 2-hr adherence step, nonadherent cells were removed, and the

plastic coverslips containing the adherent cells were covered with medium and incubated in a 5% COi environment at 37°for 24 hr. The cells

were then washed with 0.9% NaCI solution and fixed with either glutar-aldehyde or the esterase fixative. The cells were examined using phase-

contrast microscopy to determine the amount of spreading. A minimumof 200 cells was counted on each preparation. Adherence was charac

terized by the cells becoming elongated, flattened, and dark, whereasnonadherent cells appeared rounded and light under phase-contrast

microscopy (24). Spreading of normal, control macrophages ranged from70 to 80%.

Pinocytic Rate. 2.4 x 106 macrophages were incubated for 24 hr at37°on a plastic Petri dish. Nonadherent cells were removed by extensive

washing. One mg of HRP (type 2, No. P-8250; Sigma) was dissolved in

1 ml of medium and added to the adherent cells (21). Care was taken tocover all cells, which were incubated for 2 hr at 37° using sterile

techniques. After the incubation, the dish was washed extensively 9times with medium at 37°to remove all free (nonpinocytosed) HRP. The

adherent cells were then lysed with 1 ml of 0.05% Triton X-100 for 10

min. 100 n\ of the lysate were then taken, added to 900 ¡Aof thesubstrate, and transferred to 1.25-ml quartz cuvets (path length, 1 cm).The substrate for the HRP, 0.003% H2O2 and O-dianisidine (0.083 mg/

ml) (Sigma) was dissolved in 0.1 M phosphate buffer, pH 5.0. Theabsorbance was then determined at a wavelength of 460 nm. Thepinocyte rate was calculated as ng of HRP/106 macrophages/hr. Normally, 10e adherence cells pinocytose at an approximate rate of

0.0025%/hr.Binding and Phagocytosis of EA,. Sheep erythrocytes (Mission Labs,

Rosemead, CA) were incubated with a subagglutinating amount of 7SIgG rabbit anti-E (Cordis Laboratories, Miami, FL) for 30 min at 37°.The

EA, were washed 3 times and suspended in medium at 0.5% v/v.To quantitate Fc, receptor binding, the plastic adherent cells were

incubated with 0.5% (v/v) EA, for 60 min at 4°. The dish was then

washed with 0.9% NaCI solution, and the cells were fixed with 2%glutaraldehyde. Cells were stained with Giemsa (Sigma) for 15 min, and200 cells were counted to determine the percentage of adherent cellsforming EA, rosettes (>3 EA,/cell was regarded as positive) (22).

Parallel studies were performed to determine the degree of phagocytosis of EA,. Adherent cells were incubated with 0.5% EA, (v/v) for 60min at 37° and washed twice with 0.9% NaCI solution at 37°, and

nonphagocytosed cells were lysed with 1 ml of 0.85% NH4CI for 10 minat 20°.The adherent cells were then washed 3 times with normal saline

and fixed with 2% glutaraldehyde. Cells were stained similarly, and aminimum of 200 was counted to determine the phagocytic rate.

Nonspecific Latex Particle Phagocytosis. 25 n\ of 1% v/v latexparticle (diameter, 1.01 ^m) suspension in 250 n\ of medium was incubated at 37° for 60 min with 4 x 106 adherent macrophages. The

adherent cells were then washed 9 times with normal saline to removenonphagocytosed particles, fixed in 2% glutaraldehyde, and examinedunstained. The percentage of cells ingesting >5 latex particles was then

determined (22).

RESULTS

Morphological Alterations in Macrophages following Incubation with 4-HPCY. Adherent and nonadherent cells wereexamined for morphological changes by light- and phase-contrast microscopy at 2 and 24 hr after incubation with 4-HPCY.

No changes were noted at 2 hr in either the adherent or nonadherent cell populations but, at 24 hr, striking changes wereapparent in the esterase-positive adherent cell population. Few

adherent cells remained at 100 ¿¿g/ml,but those still presentexhibited marked vacuolation of the cytoplasm, best demonstrated under phase-contrast microscopy. At 4-HPCY concentra

tions between 0.5 and 10 /¿g/ml,vacuolation increased with theconcentration. Occasional cells exhibited vacuoles at 0.5 ¿tg/ml.Similar morphological changes were absent in the esterase-negative adherent or nonadherent cells over the range of 4-

HPCY tested.The gross morphological changes were confirmed by electron

microscopy. Normal-appearing, untreated adherent macro

phages at 24 hr are represented (Figs. 1 and 2). The cytoplasmic

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M. Jason et al.

vacuolization noted in the 4-HPCY-treated cells on light- andphase-contrast microscopy is well demonstrated over the range

0.5 to 10 Mg/ml and represents distension of rough endoplasmicreticulum (Figs. 3 and 4). Loss of cytoplasmic spreading andnormal endoplasmic reticulum is also noted. The mitochondria,centrioles, Golgi apparatus, and nuclear structure appear unchanged.

Effect of 4-HPCY on Viability of Adherent and Nonadherent

Cells. At 2 hr postincubation, there is no significant reduction inviability of adherent (Chart 1) or nonadherent cells. At 24 hr,there is a slight reduction in viability of the adherent cell population from a mean of 97% in controls to a mean of 91% at 4-

HPCY (0.5 Mg/ml). However, this slight reduction in viability didnot increase significantly over the range 0.5 to 10 ¿tg/ml.Viabilitywas determined in the nonadherent cell populations at 2 and 24hr (Chart 2). At 24 hr, using 4-HPCY (10 /¿g/ml),the mean

viabilities of adherent and nonadherent cells were 91 and 69%respectively. Few cells were present in the medium covering theadherent cells, possibly indicating preferential destruction of thenonadherent cells. Using 100 /¿g/mlat 2 hr, the mean viabilitiesof the adherent and nonadherent cell populations were 95 and94% respectively. However, using 100 ¿¿g/mlat 24 hr, only 30%of the nonadherent cells remained viable. Few viable adherentcells remained, but those present exhibited marked cytoplasmicvacuolization. 95% of the cells in the supernatant of the adherentcells were nonviable at 24 hr. This suggests marked toxicity of4-HPCY on all cell populations at 24 hr using a concentration of100/j.g/ml.

Effect of 4-HPCY on Adherence and Spreading of Macro-

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Chart 1. Percentage of viability of adherent mononuclear cells 2 and 24 hr afterincubation in various concentrations of 4-HPCY. Bars. S.E.

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Chart 2. Percentage of viability of nonadherent mononuclear cells 2 and 24 hrafter incubation in various concentrations of 4-HPCY. Bars, S.E.

phages. Macrophages characteristically adhere to glass andplastic surfaces, with spreading apparent at 24 hr (24, 25). Twohr after the 4-HPCY incubation (Chart 3), no significant reduction

in adherence could be demonstrated over the range 0.5 to 10/jg/ml. At 24 hr, no significant reduction in adherence could bedemonstrated up to 5 ng/ml but, at 10 /¿g/ml,there was asignificant reduction in the adherence rate which became moremarked at 100 /¿g/ml.

Spreading of adherent cells was examined at only 24 hr. Thisparameter was significantly affected at the lowest 4-HPCY concentration used, while, at >1 ¿¿g/ml,spreading was markedlyreduced or abolished, although the cells remained adherent andviable (Chart 4). This observation parallels the lack of plasmamembrane spreading demonstrated by electron microscopy.

Effect of 4-HPCY on Pinocytosis of HRP. The pinocyticactivity of adherent macrophages was studied at 24 hr only. Amarked reduction in the uptake of HRP was demonstrated at 4-HPCY concentrations >0.5 //g/ml (Chart 5). This effect wasnoted at 4-HPCY concentrations which also affected spreading,

again suggesting a functional defect in the macrophage cytoplasmic membrane at lower 4-HPCY levels.

Effect of 4-HPCY on the Binding of EA to Fc Receptors.

The ability of adherent M0 to bind EAT by their Fa, receptorswas measured 2 hr after preincubation with 4-HPCY. It shouldbe noted that binding is significantly reduced at 4-HPCY concentrations >1 itg/ml (Chart 6) but not at 0.5 /ig/ml. Reduction inphagocytosis mediated by Fc, binding at 2 hr (Chart 7) parallelsthe reduction in the Fc binding.

Effect of 4-HPCY on Nonspecific Latex Particle Phagocy-

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Chart 3. Percentage of adherent mononuclear cells at 2 and 24 hr after incubation in various concentrations of 4-HPCY. Bars, S.E.

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Chart 4. Percentage of spreading of adherent mononuclear cells at 24 hr afterincubation in various concentrations of 4-HPCY. Bars, S.E.

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Macrophages and CY

260

210

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Charts. Pinocytosis of horseradish peroxidase by adherent mononudear cellsat 24 hr after incubation in various concentrations of 4-HPCY. Bars, S.E.

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Chart 6. Percentage of adherent mononudear cells demonstrating Fc, surfacebinding of EA, at 2 hr after incubation in various concentrations of 4-HPCY. Bars,

S.E.

tosis. To ascertain whether the phagocytic defect resided inreduced Fa, receptor production or an abnormality in membranefluidity, the effect of 4-HPCY on nonspecific latex particle phag

ocytosis was studied. A similar reduction in latex particle uptakewas demonstrated at 2 hr (Chart 8) at 4-HPCY concentrations>0.5 /tg/ml.

DISCUSSION

The aim of this study was to determine if the activated metabolite of CY, in concentrations and concentration x time (C x T)exposure which might be achieved in serum by chronic low-dose

administration (<2 mg/kg/day), had any effects on the adherent,esterase-positive, mononudear cell population (macrophages)

derived from human peripheral blood. To study the in vitro effectsof CY, the analogue 4-HPCY was used, which is spontaneouslyhydrolyzed to 4-HCY (12). These data demonstrate profound

and reproducible effects of CY on isolated macrophages, bothon structure, as demonstrated by light, phase, and electronmicroscopy, and on function, as demonstrated by adherence,spreading, Fa, receptor binding, phagocytosis (specific Fa, receptor and nonspecific latex particle), and pinocytosis. Theseeffects are observed with CY concentrations at which viability ofthe cell population is maintained and at which DNA cross-linkinghas not been detected in drug-sensitive tumor cells (4).

The morphological changes present at 24 hr consist of reducedmacrophages spreading and the development of dose-related

intracytoplasmic vacuoles. By electron microscopy, these

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Chart 7. Percentage of adherent mononudear cells demonstrating phagocytosisof EA, at 2 hr after incubation of the cells in various concentrations of 4-HPCY.

Bars, S.E.

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Chart 8. Percentage of adherent mononudear cells demonstrating phagocytosisof latex particles at 2 hr after incubation in various concentrations of 4-HPCY. Bars,

S.E.

changes are reflected within the cytoplasm by gross dilataion of,and reduction in, the quantity of HER and marked reduction inmembrane spreading. No gross changes are present within thenucleus, Golgi apparatus, or mitochondria. With respect to function, CY in the concentration range of 0.5 toi 0 ng/m\ results inimpaired Fa, receptor binding, reduced phagocytosis of EA^ andlatex particles, and reduced pinocytosis, indicating impaired receptor and membrane function. Unlike the effects of gold onmacrophages, which result in vacuoles due to the formation ofgiant phagolysosomes (25), the vacuoles produced by CY represent dilatation of the PER, the site of protein synthesis withinthe cell. If the material within the RER represents protein, itsuggests that: (a) following protein synthesis, there is an inabilityto transport protein to the Golgi apparatus; and/or (o) proteinsynthesis is inhibited directly with accumulation of t-RNA-amino

acids or incomplete polypeptide fragments within the vacuole.As the macrophage is intimately involved with antigen in the

immune response, CY metabolite alteration of the macrophagemight alter the immune response at several sites, including (a)uptake, processing, and presentation of antigen to T-lymphc-cytes; (b) macrophage-T-lymphocyte interaction; (c) productionof interieukin 1, necessary for the proliferation of T-helper lymphocytes and B-lymphcoytes, respectively; and (d) the effector

role that the macrophage plays in the control of infectious agentsand tumor cells (17, 18, 20, 26).

While there is a definite in vitro effect of 4-HPCY on isolated,

peripheral blood, adherent, mononudear phagocytic cells, this

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M. Jason et al.

study has certain limitations: (a) it does not address the in vivoeffects of CY, although several studies (5, 23) have suggestedthat CY acts via a direct effect on lymphocytes; and (b) itconsiders only a mononuclear phagocytic cell population withinperipheral blood and does not consider the effect of the drug oncommitted and differentiated fixed tissue macrophages.

In these studies, significant effects on macrophages were seenat concentrations of 4-HPCY of 0.5 to 1.0 tig/m\ or 1.7 to 3.4nmol/ml. The total drug exposure (C x 7) of the macrophagesto 4-HPCY was 1.7 to 3.4 nmol/ml. Pharmacokinetic studies byWagner ef al. (27) have shown peak 4-HCY levels of 1.37 ^g/ml

or 4.7 nmol/ml and C x T exposure of 16.7 nmol/ml hr after ani.v. dose of CY of 20 mg/kg. The lowest doses of CY usuallyused in antitumor and immunosuppressive studies have been inthe range of 2 mg/kg. Studies of 4-HCY levels after such low

doses of CY have not been carried out. However, measurementsof the parent compound and another metabolite, phosphoramidemustard, after different doses of CY, have shown what appearsto be a linear relationship between the dose of CY and peakplasma levels and C x 7 exposure of these compounds. If sucha dose-drug level relationship holds for CY and 4-HCY, then adose of CY (2 mg/kg) should produce a peak plasma level of 4-

HCY of about 0.14 M9/ml (0.47 nmol/ml) and a C x 7 exposureof approximately 1.6 nmol/ml hr. Since CY is well absorbed afterp.o. administration (about 90%) (13), the clinical route of administration should not affect our estimates. Thus, concentrationsof 4-HPCY shown to be effective in these studies are similar to

those which are present in plasma after administration of evenlow doses of CY. In particular, the total drug exposure (C x 7)of macrophages to 4-HPCY should be very similar to the exposure to 4-HCY produced by the administration of CY (2 mg/kg).

Previous in vitro studies using 4-HPCY have focused on human

(19) and murine (6, 15) lymphocyte subpopulations in variousfunctional assays. Using macrophage-depleted (<2%) cell populations, Ozer ef al. 919) demonstrated that human presuppres-sor cells for B-effector function which differentiate in responseto Con A are extremely sensitive to low concentrations of 4-HPCY. While the lowest 4-HPCY concentration they used for

presuppressor sensitivity was significantly lower than that usedin this study (3.3 x 10~3 versus 0.5 ^g/ml), isolated macrophages

clearly underwent morphological and functional changes to alevel of 0.5 /ig/ml [5 /¿g/mlis necessary for detectable DMAcross-linking in L1210 cells (4)]. Macrophages were markedlyaffected by 4-HPCY concentrations which would have resultedin loss of the T-inducer (helper) cell populations or differentiatedT-suppressor cells (19).

Our studies have indicated that activated CY, in a concentration range commonly achieved in vivo, results in profound effectson peripheral blood mononuclear phagocytic cell morphologyand function. If these changes occur in vivo, it is possible thatsignificant immunosuppression produced by CY may result froman effect of CY on the macrophages, at activated CY levelswhich do not produce cross-linking of DMA in sensitive cells.

Further functional studies are underway to analyze the effectsof CY on the macrophage in its role as an accessory cell in bothantigen-specific and antigen-nonspecific blastogenic assays.

ACKNOWLEDGMENTSThe authors wish to thank Nell Greco and Linda Walter for their help in preparing

the manuscript and Gary Mirick and Dr. Monique Berman for their technical adviceand assistance.

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3940 CANCER RESEARCH VOL. 44

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Fig. 1. EM study of adherent untreated mononuclear cells at 24 hr. Note the prominent plasma membrane extensions, the normal nuclear structure with a loosechromatic pattern, and the formation of abundant small intracytopiasmic inclusions representing normal cytoplasmic granules. Original magnification, x 1900.

Fig. 2. EM study of an adherent untreated mononuclear cell at 24 hr. Note diffuse normal-appearing rough endoplasmic reticulum with abundant ribosomes. Original

magnification, x 4900.Fig. 3. EM study of an adherent mononuclear cell 24 hr after a 1-hr incubation in 4-HPCY (1.0 ng/ml). Note the presence of numerous large vacuoles due to dilatation

of the rough endoplasmic reticulum (arrows), the loss of normal rough endoplasmic reticulum, and the absence of plasma membrane spreading but preservation of thenormal nuclear structure. Original magnification, x 9000.

Fig. 4. EM study of an adherent mononuclear cell 24 hr after a 1-hr incubation with 4-HPCY (1.0 jig/ml). Note marked distension of rough endoplasmic reticulum(arrow) with ribosomes lining the vacuole. Original magnification, x 9000.

SEPTEMBER 1984 3941

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1984;44:3936-3941. Cancer Res   Mark Jason, Brian S. Andrews, Michael Colvin, et al.   Mononuclear Phagocytic Cells (Macrophages)Morphology and Function of Human Peripheral Blood

Effects of 4-Hydroperoxycyclophosphamide on theIn Vitro

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