Effects of treatment with immunomodulatory drugs on thymus and spleen lymphocyte subpopulations and serum corticosterone levels

Effects of Treatment with lmmunomodulatory Drugs on Thymus and Spleen Lymphocyte Subpopulations and Serum Corticosterone Levels Patricia E. Fast, Cheryl A. Hatfield, Cheryl L. Franz, Earl G. Adams, Norman J. Licht, and Margaret V. Merritt Abstract: Immunofluorescence was used to charactenze the lymphocyte subpopulatzons of mice treated with six immunomodulatory drugs hydrocorttsone acetate (HCA), cortzcosterone acetate (cortzcosterone), cyclophosphamlde, cytosine arabznoszde (Ara-C), 15(S)-methyl prostaglandln E 1 (15(S)-methyl PGE1), and 2-ammo-5-bromo-6-phenyl-4-(3H)-pynmzdmone (ABPP) The number of thymus and spleen cells beanng Thy-1, Ig, Lyt-1 and Lyt-2 antigens and the density of the antigens on each cell (IF profiles) were determined Microscopic examination of cells stained with rhodamme-labeled antI-Lyt-2 and fluoresceln-labled anti-Lyt-1 was used to measure the propo~on of Lyt-1 +2-, Lyt-1 +2 +, and Lyt-1-2 + cells in the spleen and thymus of drug-treated animals The changes In lymphocyte subpopulatlons were compared with the vaned effects of these drugs on antibody formation and graft vs host (GVI-I) reaction Three immunosuppresswe drugs, HCA, cyclophosphamzde, and Ara-C, depleted the thymus of cells expressing a large quantity of Thy-1 The drug-resistant cells were larger and had more Lyt-1 than ceils from control animals HCA treatment depleted the thymus of Lyt-1 +2 + cells, the cortisone resistant cells were primarily Lyt-1 +2- Cyclophospham~de and the antwzral lmmunostlmulant, ABPP, caused szmdar, but less marked, alterations The proportion of Lyt-1-2 + ceils in the thymus was reduced by treatment with all the drugs, but the density of Lyt-2 on the drug-resistant ceils was not altered Treatment wlth Ara-C or 15(S)-methyl PGE 1 produced a very modest evaluation m Lyt-l+2 - cells 15(S)-Methyl PGEI, which suppresses some Immuno-lnflammatory reactions, had no discernible effect on thymocyte size or the IF profile of Thy-1, Lyt-1, or Lyt-2 In the spleen, the amount of Thy-1 and of lmmunoglobuhn on cells beanng these markers was changed very little by drug treatment The proportion of splenic B cells was dlmzmshed by treatment with cyclophosphamlde and, to a lesser extent, by HCA, while the proportion of spleen ceils beanng detectable Thy-1 and Lyt-1 increased correspondingly The propo~on of cells beanng Lyt-2 was altered by only two drugs cyclophosphamlde lncrea.~_.d both Lyt-1 +2 + and Lyt-1-2 + spleen ceils and ABPP (an interferon inducer which stlmulates antibody formation) decreased both Lyt-2 + subpopulatlons Treatment with two drugs caused

Present Addresses (P E F) c/o SMAHEC, College of Human Medlcme, M1chlgan State Umverslty, Borgess Hospltal, Kalamazoo, MI 49001, (M V M ) Department of Chemlstry, Wellesley College, Wellesley, MA 02181 From Hypersensltivlty Diseases Research (P F, C A H ), Physical and Analybcal Chemlstry (C L F, N J L, M V M ), and Cancer Research (E G A ), The Up]ohn Company, Kalamazoo, Mlchlgan Recelved October 8, 1981, accepted July 14, 1982 Address requests for reprints to Cheryl A Hatfleld, Hypersenslhwty Diseases Research, The Up)ohn Company, Kalamazoo, MI 49001

~) ElsewerSaence Pubhshmg Co, Inc, 1982 52 Vanderbflt Ave, New York, N Y Immunopharmacology5, 135-155 (1982)

13S 0162-3109/82/0613521502 75

136

P E Fast et al the serum co~costerone concentration to nse ABPP increased serum corticosterone substantially while the prostaglandin induced a smaller and more tmnsttory increase An indirect mechanzsm, via cortlcosteroid release, might explain the thymlc depletion observed ~n mice treated with 15(S)-methyl PGE 1 and ABPP, but neither the suppresszon of the GVH reaction by these drugs nor polyclonal actwatlon of B cells by ABPP can be attnbuted to endogenous corttcosteroids Our data show that enumeration of splemc lymphocyte subpopulatzons by immunofluorescence techmques may aid zn eluod~zng the mode of action o/ immunomodulatory drugs

Key Words: Immunomodulatory drugs, Flow cytometry, Lyt-l, Lyt-2, Serum cortacosterone levels, Splenocytes, Thymocytes, Thy-1

INTRODUCTION The discovery of dlfferences in expresslon of various surface antigens by mouse lymphoid subpopulations and the development of antibodies to these antigens made possible the definition of several lymphocyte populations Thy-1 is a marker for thymus-derived cells (Raft, 1969) The Lyt surface antigens identify functional T-cell populations (Cantor and Boyse, 1977) cells of the helper-amphfier subclass bear Lyt-1 but not Lyt-2 (Lyt-l+2 - cells), those of the suppressor-cytotoxic subclass express Lyt-2 but not Lyt-1 (Lyt-l-2 + cells) A third major T cell subclass, which bears both antigens (Lyt-l+2 + cells) is revolved in feedback suppression of humoral responses (Cantor and Gershon, 1979; Calkins et al, 1980) The thymus contains all three subclasses and cells differentiate before they leave the thymus (Scollay et al, 1978, Mathieson et al, 1979) Previous studles have related the proportions of lymphoid subpopulatlons, as determined by these surface antigens to immunologic functlon For example, Kalland (1980) found that mlce immunosuppressed by h'eatment with diethylstilbestrol as neonates had more Lyt-l+2 + cells and fewer Lyt-l+2 - cells in the spleen than the controls Alterations in helper and cytotoxlc suppressor subclasses of human blood lymphocytes have been related to the immune status of the individual (Reinherz et al, 1979) Mathieson et al (1979) and Ledbetter et al (1980) used flow cytometry to determine the slze and Lyt antigen expression of cortisone-reslstant and cortisone-sensitive thymocytes We report here the results of an immunofluorescence study of the effects of a variety of immunomodulatory drugs on munne lymphoid subpopulations defined by cell size and antigenic markers We have reported the effects of these drugs on membrane fluidity, determined by spin labeling, and on lipid composition of spleen and thymus cells (Merritt et al, 1982) HCA causes severe lymphoid depletion in rodents (Claman, 1972) Mature lymphocytes are less susceptible than immature thymocytes (Cohen et al, 1970) Since humans are less susceptible than rodents to corticosteroids (Ganong, 1979), it is important, when evaluating an immunomodulatory drug in rodents, to determine whether its effects may be mediated by release of endogeneous corticosteroids The relationship is not simple, though, since ~mmune responses can induce co~costerone production (Besedovsky and Sorkm, 1977, Besedovsky et al, 1981, Riley, 1981) To evaluate the possible role of co~costeroids in the action of these drugs, we measured serum corticosterone after drug administration and compared the effects of

Abbreviations. ABPP" 2-ammo-5-bromo-6-phenyl-4-(3H)-pynmldmone, Ara-C cytosine arabmoslde,

B6CBF 1 mlce (C57BI/6 × CBA)FI hybnd mlce, FACS fluorescence-actlvated cell sorter, GVH graft vs host, HCA: hydroco~sone acetate, HEPES N-2-hydroxyethylplperazme-N"-2-ethane sulfomc aod, IF imrnunofluorescence, Ig immunoglobulin, 15(S)-methyl PGEI 15(S)-methyl prostaglandin El, PFC plaque-forming cells, SRBC sheep red blood cells

Drug Effects on Lymphocytes and Serum Corticosterone

137

corticosterone and HCA on thymus and spleen cell number, graft vs host reaction, spontaneous antibody~formmg cells and the immune response to rejected SRBC.

MATERIALS AND METHODS Mice and Drug Treatment Female CBA and (C57BI/6×CBA) FI mlce, called B6CBFI mlce, were obtained from Cumberland View Farms, Chnton, TN or Jackson Laboratories, Bar Harbor, ME In each flow cytometry or comcosterone experiment, groups of at least 5 CBA mice were treated with hydroco~sone acetate (HCA), corticosterone acetate (corticosterone), cytosine arabmoside (Ara-C), cyclophosphamide, 15(S)-methyl-prostaglandm E I (15(S)-methyl PGE0, or 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP) Corticosterone was obtained from Sigma Chemical Co (St Louis, MO) and cyclophosphamlde from Falrfield Chemlcal (Blythewood, SC) or Mead Johnson (Evansville, IN) All other drugs were syntheslzed at The Uplohn Company (Kalamazoo, MI) Drug suspenslons or solutions were prepared for each experiment by m ~ n g a welghed amount of drug with saline or with saline containing 0 1% Tween 80, except the 15(S)-methyl PGEI, whlch was dlssolved m 95% ethanol and diluted with 11 5 volumes of sahne just pnor to oral adminlstration The doses and routes of admmlstratlon for each drug, indlcated in the Figures and Tables, were selected to be immunoregulatory and nontoxlc Control animals in each experiment were rejected with equal volumes of sahne or 0 1% Tween 80 m saline

Preparation of Cell Suspension Mice were killed by CO 2 inhalation and thelr thymuses and spleens placed immediately m ice-cold Eagle's Mmlmal Essential Medmm buffered wlth 25 mM HEPES and containing 5% fetal calf serum (GIBCO, Grand Island, NY), this medlum was used in all expenments unless otherv~se indicated Fascia and connective tissue were removed and single cell suspensions were prepared from each organ pool as described previously (Merritt et aL, 1982) For immunofluorescent staining, red blood cells were removed from the spleen cell preparation by NH4CI lysls (Shortman et a l , 1972), the suspensions were filtered through nylon mesh (Nitex®, 110/~m pore size, Tetko, Elmsford, NY) and viable nucleated cells (those that excluded 0 05% trypan blue) were counted using a hemacytometer

Immunofluorescent Staining For dlrect staining, fluorescem-labeled monoclonal antibody speclfic for Thy-1 2 was purchased from New England Nuclear (Boston, MA) and fluorescem-labeled monoclonal rat anti-mouse Lyt-1 and anti-mouse Lyt-2 were purchased from Becton Dickinson (Sunnyvale, CA) For indirect staining of the thymus, mouse monoclonal Thy-I 2, Lyt-1 1 and Lyt-2 1 (all from New England Nuclear) were used, followed by fluorescein-labeled goat anti-mouse IgG specific for heavy and hght chains (Cappel Laboratories, Cochranville, PA), the same anti-lg was used to label B cells m the spleen Spleen and thymus cells were stained for Thy-1, Lyt-1, Lyt-2 or Ig as follows 1 × 106 cells were incubated with 0 1 ml of antibody in medium at a saturating concentration (dilutlons are indicated in the Figure legends) for 45-60 mm on ~ce. The ratlo of antiserum to cells was carefully controlled to make posslble the quantitative comparison of antigen expressed on cells from different groups After incubation, the cells were washed three times by centrifugation at 4°C The staining procedure was repeated for md~rect staining In

138

P E Fast et al

some experiments, cells were stained for two antigens simultaneously by incubating them with one fluorescein-labeled antibody and one biotin-labeled antibody followed by rhodaminelabeled avldin (all from Becton Dickinson, Sunnyvale, CA), using the procedures described above. For double-labeling experiments, 0 I% sodium azlde was added to the medlum, and the fetal calf serum was reduced to 2% The cells stained with fluoresceinated antlbody were either examined by flow cytometry within a few hours or fixed by addilion of an equal volume of 2% paraformaldehyde buffered vvlth 0 01 M phosphate and stored in the cold for later analysls Fixatlon reduced the overall fluorescence intensity, but altered neither the proportion nor the relative intensity of the stained cells (unpublished data). In expenments using double-labeling, the fixed cells were examined using a Leitz epi-illummated fluorescence microscope to determine the number of green, red, unstained, and two-colored cells, dead cells, distinguished by umform intracellular staining, were dlsregarded

Flow Cytometry Thymus and spleen cells were examined using a fluorescence-activated cell sorter (FACS II, Becton Dickinson FACS Systems, Sunnyvale, CA) The instrument and its apphcatlon have been described (Herzenberg and Herzenberg, 1978) At least 3 × 104 nucleated cells from mice treated with drugs were analyzed for cell size and distribution of surface antlgens In all cases, fluorescence was determined for vlable lymphocytes by setting electronic gates to exclude measurements from non-viable cells and residual red blood cells or platelets The peaks containing viable cells were identified by staining unfixed, unlabeled cells with 25/~g fluorescem dlacetate (Rotman and Papermaster, 1966) The histograms depicting fluorescence intensity vs number of cells are called immunofluorescence (IF) profiles Because the dlstnbution of antigen on spleen cells was so heterogeneous, IF profiles were acqulred using a Iogarithmlc amplifier, whlch prevented the loss of data for the dimmest cells Replicate flow cytometnc analysis of the same stained spleen cell sample gave a fluorescence peak position that vaned by only one channel in 256, while the peak positions of samples from several mice did not vary more than 3 channels The variation in percent cells stained in samples from individual mice (4%) was greater than m replicate samples from the same mouse (1%) Therefore, we concluded that most of the variatlon between experiments was due to differences between the mice rather than the staining techniques or FACS analysis In assessing drug effects we consldered a change of 8% or greater significant (twice the mouse-to-mouse vanations)

Antibody Response Sheep erythrocytes (SRBC) in Alsever's solution (Colorado Serum Company, Denver, CO) were washed three tlmes m phosphate-buffered sahne (0 001M, pH 7 4), 1 × 108 SRBC were injected intravenously into CBA or B6CBF I mlce on day 0 Mice were treated with immunomodulatory drugs as indicated in Figure 1 and direct plaque-formlng cells (PFC) in the spleen were measured on day 4 PFC were detected by a slide modlficatlon (M1shell and Dutton, 1967, Plotz et al, 1968) of the hemolytic PFC assay of Jerne and Nordm (1963) In addition, ummmumzed mlce were treated with the drugs to assess the number of cells makmg spontaneous (background) antibody specfic for SRBC For slmphc~ty, we deslgnate the day of assay as day 4 Late Phase Graft vs Host (GVH) Assay B6CBF I recipient mice (GVH trace) were injected intravenously wlth 1 × 108 vlable CBA spleen cells on day 0 (Howard and Woodruff, 1961) Control B6CBF I mice (non-GVH mice) were not injected wlth splenocytes. (We had previously established that untreated anlmals were identlcal

Drug Effects on Lymphocytes and Serum Co~costerone

139

as non-GVH controls to those rejected with 1 × 108 syngene~c splenocytes ) Groups of 5 - 1 2 F I mlce that had been injected with parental spleen cells to induce GVH and non-GVH FI controls of the same age and sex were treated with the drugs on days 7 - 9 of the GVH reaction On day 10, the recipient m~ce were k~lled and their spleens were removed, tnmmed, blotted and weighed The ratio of spleen weights from drug-treated mice to saline controls was determined separately for GVH m~ce and non-GVH m~ce to determine two things first, whether each drug affected spleen weights in GVH mice, and second, whether the effects were selective for GVH or were also seen m non~GVH mice The data were not combined into one index, since at least one drug has qualitatavely dxfferent effects on GVH and non-GVH spleen weights

Corticosterone Determination Serum co~costerone concentrations were determined by radioimmunoassay using kits from Radloassay Systems Laboratones, Inc (Carson, CA). Serum samples were stored at 4°C and diluted 200-fold with assay buffer pnor to analysls in duplicate The antibody used m the co~costerone radiolmmunoassay cross-reacted 100% with co.sol Serum cortisol levels were therefore determined by radlolmmunoassay using Quantimune® Cortisol RIA klts (Blo-Rad Laboratories, Richmond CA) in some samples to determine the contributlon of cortlsol to the co~costerone assay The antlbody directed against cortisol had only a 3 7% cross-reactiwty w~th cortlcosterone When mlce were injected wlth HCA, the cortisol and "cortlcosterone" measurements were comparable, as expected Animals treated w~th e~ther hydrocortisone or corticosterone were used as positive controls m the corticostero~d experiments Untreated mlce or m~ce injected intraperitoneally (l p ) with 0 2 ml of sahne were included as negative controls to reflect the effects of stress and diurnal variatlon m each expenment Nolse and cage handhng were avolded before and during the expenment to avold hlgh endogenous levels of corticosterone

RESULTS Effects of the Drugs on the Antibody Response and GVH Reaction Figure 1 shows that the immunomodulatory drugs under mvestlgatlon had very diverse effects on the primary antibody response to sheep erythrocytes Treatment with HCA one day after ~mmunizataon suppressed the response while corticosterone had no slgnificant effect Treatment wlth either drug on the day of immumzation or wlth HCA two days before immumzatlon dlmmlshed the antlbody response only slightly (data not shown) Cyclophosphamlde and Ara-C virtually abolished the antibody response, whereas 15(S)-methyl PGE I had no effect ABPR on the other hand, was a strong adjuvant In addihon, ABPP treatment caused a 10 to 100-fold increase in background anUbody-formmg cells m the spleens of unimmunized mlce (Fast and Stnngfellow, 1980, see also Table 6) Figure 2 shows the effects of these drugs on spleen weights of F I non-GVH mlce and GVH mice, the drugs were glven late m the GVH reaction In general, the late phase of the GVH reactlon can be regarded as a model ~mmuno-mflammatory reaction mxtiated by two cooperatmg T cell subsets (Cantor and Asofsky, 1975) Only two of the drugs had a selectlve effect on the GVH spleen welghts 15(S)-methyl PGE 1 mhlblted GVH splenomegaly but dld not significantly alter the spleen welghts of non-GVH mlce Thls selective effect was confirmed m slx addltlonal expenments (data not shown) Paradoxlcally, ABPP reduced the GVH reactlon although It increased spleen welght of non-GVH control mice Thls effect of 500 mg/kg/day ABPP was not statlstlcally slgnificant, and the drug was toxlc at thls dose m GVH m~ce (only), as evldenced by weight loss and some mortahty, therefore, the expenment was repeated using 250 mg/kg/day ABPP The mean welght (_+ standard devlatlon) of GVH spleens from drug-treated

125 250

;orticosterone =yclophosphamide

500

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1,2,3

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1

1

Time (day)

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I

I ~O

100

:)

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200

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300

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400

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500

PFC/Spleen (Percent of Control)

:igure 1 Effects of immunornodulatory drugs on the primary antzbody response to SRBC Mice were mmumzed day O, treated as indicated, and thelr spleens were assayed for direct PFC on day 4 The data are the "atlos of the means of triplicate determznatlons on each spleen uslng groups of at least 5 mice The asterisks ndzcate those values that are slgniflcantly different from those for the saline-treated controls by the t test, *, p <~ ) 05 and **, p ~ 0 01 The values reported for 15(S)-methyl-PGE I andABPP are the means of 3 experiments

~BPP

15(S)-Me-PGE1

50

125

1CA

~,ra-C

Dose (mg/kg/day)

)rug

i--i

500

2 7,8,9

7,8,9

7,8,9

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7

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Figure 2 Effects of immunomodulatory drugs on the late phase of the GVH reactzon FI mzce were uninjected (non-GVH) or injected wzth parental spleen cells (GVH) on day 0 and treated days 7 - 9 wzth drug or sahne, their spleens were wezghed on day 10 Left column mean spleen weight non-OVH F I mice treated wzth drug~mean spleen wezght non-OVH FI mzce treated wlth saline Rzght column mean spleen welght GVH drug-treated mlce/mean spleen wezght GVH sahne-treated mice. Data from several expenments are zncluded, in each, the mean spleen wezght of the GVH-sahne treated animals was 2 - 3 t~mes that of the non-GVH saline controls The close of Ara-C, 30 mg/kg/day, was shghtly dlfferent than that used zn the other expenments of th~s report (50 mg/kg/day) The results were evaluated by the t test **,p 4 0 0 1

ABPP

15(S)-Me-PGE 1

30

250

Cyclophosphamide

Ara-C

125

Dose Time (mg/kg/day) (day)

Corticosterone

Drug

Spleen Weight Ratio (Day 10) of Drug-Treated / Saline-Treated Mice

142

P E Fastetal

mlce was 199 _+ 26 mg, compared to 255 _+ 31 mg for sahne-treated GVH mlce (p ~< 0 01) The non-GVH spleen weights were increased from 73 _+ 9 to 114 _+ 14 mg by treatment with ABPP (p ~< 0 001) In a second experiment, GVH spleen welghts were 113 _+ 19 mg m ABPP-treated mlce and 179 -- 17 mg m saline-treated (p ~ 0 01), but non-GVH spleen weights were not significantlyincreased (65 _+ 8 vs 61 -+ 11 mg) The other drugs lacked selechve effects on GVH Cyclophosphamlde reduced spleen welghts of both control and GVH mlce, HCA slgnificantly reduced non-GVH spleen welghts only, while corticosterone and Ara-C had no sigmflcant effects (Figure 2) In other experiments (not shown), both Ara-C and cyclophosphamlde inhibited the GVH reaction when glven during the early and mlddle phases (days 1 - 3 and 4 - 6 , respectively)

Cell Number and Cell Size Treatment wlth any of the compounds reduced the number of viable cells recovered from the spleen and thymus, HCA, cyclophosphamlde, and Ara-C caused the greatest reduction (Table I) Spleen weights were decreased, in a fashlon roughly proportional to cell loss, by each of the compounds except ABPP Treatment wlth ABPP produced enlarged spleens but still caused a loss m recoverable viable cells The predominant cells in the thymus of sahne-treated m~ce were small (peak at channel 96 m the light scatter histograms obtained on the FACS), larger cells made up a very small fraction of the normal thymus (data not shown) After treatment with HCA, Ara-C, ABPP, or cyclophosphamide, larger cells (peak at 114) were prominent Treatment of mlce wlth 15(S)-methyl PGE 1 dld not alter the slze dlstnbutlon of cells m the thymus None of the drugs tested caused a slgmficant alteratlon m the slze distribution of spleen cells (data not shown)

Thymocyte Subpopulations Thymus cells were stained dlrectly wlth fluorescemated antl-Thy-1 2 Essentially, all of the thymocytes had Thy-1 on their surfaces but the quantlty was quite vanable Treatment w~th ABPP reduced the number of cells wlth a large amount of Thy-1 (top panel of Figure 3) A slmilar decrease was seen after treatment wlth cyclophospham~de and Ara-C and to a greater extent after treatment wth HCA (Table 2) An alteratlon of 8% or greater was considered

Table I

The number of vzable nucleated cells recovered from the thymus and spleen of drug-treated rmce

Drug HCA Co~costerone Cyclophosphamlde Ara C 15(S)-methylPGE I ABPP

Dose (mg/kg/day)

Drug Treatment a

125 125 250 50 2 500

2 2 I 1,2,3 1,2,3 1,2,3

Mean Number of Viable Cellsb (% of Safine Control) Thymus Spleen 10 23 5 9 53 30

(3-28) (15-31) (3-6) (8-10) (36-67) (11-47)

63 85 3 58 78 68

(42-81) (74-106) (2-3) (30-74) (36-100) (48-87)

aDay or days dosed relatlveto assay on day 4 15(S)-MethylPGE1was admmlstered orally, the other drugs were rejected ~ntrapentoneally bMean (range) for 3 - 5 experiments expressed as percent of recovery for sahne-treated controls The mean • number of vlable cells recovered from sahne-treated m~cewas 6 8 × 107 per thymus (range 5 I - 13 7) and 5 1 x 107 per spleen (range 3 7 - 9 7) V~abdltyfor cellsfrom thymus and spleen from sahne-treated controls was >83% and :>78%, respec~vely,m these expenments In smallerp11otexpenments whlch were performed more qulckly,slmllardrug effectswere seen and vlabdltywas greater (~95% for thymus and ~90% for spleen cells )

143

Drug Effects on Lymphocytes and Serum Corticosterone

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80 127

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255

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80 127

255

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l

15 45 105 255 15 45 105 255 Fluorescence Intensity (Arbitrary Units)

Figure 3 The scatter-gated fluorescence hlstograms of thymocytes from saline and ABPPtreated mice Top panel thymocytes stained to detect Thy-1 The cells were stained with a 1/100 dilution of fluorescemated anti-Thy-1 2 using the procedure descnbed In the text Cells incubated with medium only were detected in channels 1 - 1 0 Middle panel thymocytes stained to detect Lyt-1 The cells were stained wlth a 1/50 dilution of anti-Lyt-1 I followed by a 1/40 dilution of fluoresceinated goat antl-mouse IgG using the procedure descnbed In the text Cells incubated with medium only were detected In channels 1 - 1 0 Bottom panel thymocytes stained to detect Lyt-2 The cells were stained wlth a 1/250 dilution of antt-Lyt-2 1 followed by a 1/40 dilution of fluorescemated goat antl-mouse IgG using the procedure described in the text Cells incubated wlth medium only were detected in channels 1 - 1 5 significant (see Methods) Treatment with 15(S)-methyl PGEt or corttcosterone had little or no effect Exammatlon of the dot plots, which dlsplay fluorescence intensity of a smgle cell as a funchon of the slze of that cell, confirmed that the larger cells remaining m the thymus after treatment with HCA+ cyclophosphamtde, Ara-C or ABPP had httle Thy-1 on their surfaces (data not shown) Nearly all the thymocytes (~95%) from all treatment groups expressed some Lyt-1 The mlce treated wlth drugs other than the prostaglandm had more thymocytes w~th a large amount of Lyt-1 (Table 2) Typical IF profiles of thymus cells from saline- or ABPP-treated mlce, stained mdlrectly with anti-Lyt-1 I, are shown m the center panel of Figure 3 Cells remaining m the thymus after treatment wlth HCA or cyclophosphamlde had more Lyt-1 on each cell (the peak of the IF profile was at channel 127) than those from mlce treated with Ara-C or ABPP (peaks at 72 and 56, respechvely) The peak intensity for cells from mlce treated wth sahne was at channel 37, treatment with comcosterone or 15(S)-methyl PGE I had little effect on the IF profile

144

Table 2

P E Fast et al

Antzgen expression on t h y m o c y t e s f r o m drug-treated a mice

Drug

% Thy-1 b

% Lyt-1 c

% Lyt-2 ~

Bright

Bnght

Positwe

91 31 95 45 49 82 59

33 84 49 78 70 36 70

84 37 79 44 48 70 51

Sahne HCA Co~costerone Cyclophosphamide Ara C 15(S)-methyl PGE 1 ABPP

aThe drug doses and tlmes of treatment are descnbed in Table 1 The values shown are from 1 - 3 expenments, results from different expenments agreed within a few percent for all treatment groups The range of values obtained for the sahne controls Thy-1, 88-95%, Lyt-1, 32-34%, and Lyt-2, 83-86% bCells bnghfly stained wth fluorescemated ant1-Thy-12 as detected by flow cytometry (Allthymocytes m these expenments expressed some Thy-1, bright cells had a fluorescence mtenslty > channel 76 See Figure 31 CCellsbnghtly stained wlth fluorescematedant1-Lyt-1 (Allthymocytes m these experiments expressed some Lyt-1, bright cells had a fluorescence mtenslty > channel 80 See Figure 3) aCells stained wlth an~-Lyt-2 (channels > 45, see Figure 3) Some thymocytes bore no detectable Lyt-2

Not all thymocytes bore detectable Lyt-2 (Table 2) Treatment with all of the compounds except co~costerone reduced the number of cells expressing Lyt-2, HCA and cyclophosphamide caused the greatest reduction and the prostaglandin caused the smallest reduction The bottom panel of Figure 3 shows typlcal IF profiles of thymus cells from ABPP- and saline-treated mice stained mdlrectly for Lyt-2 The hlstograms of thymocytes from ammals treated with HCA, cyclophosphamide, or Ara-C were slmilar to that shown for ABPP, while the IF profiles from thymocytes from mlce treated v~th 15(S)-methyl PGE I or with corticosterone more closely resembled that of the saline group The p r o p o ~ o n of cells beanng only Lyt-1 (Lyt-l+2-), only Lyt-2 (Lyt-l-2+), or both antigens (Lyt-l+2 +) was determined by double immunofluorescence staining (Figure 4) The thymus of the sahne controls contained pnmanly Lyt-l+2 ÷ cells, whereas the spleen contained more Lyt-l+2 - cells All drugs decreased the Lyt-l-2 + thymocyte subpopulation HCA treatment markedly reduced the proportion of Lyt-l+2 + cells m the thymus, cyclophosphamlde and ABPP produced simdar but more modest changes The sums of Lyt-I + and Lyt-2 + cells counted using the microscope agreed with the percentages we determined by flow cytometry m the same experiment and separate expenments

Lymphocyte Subpopulations in the Spleen Spleen cells were stained dlrectly for Thy-l, Lyt-l, Lyt-2 and Ig (Figure 5) A few more cells wth surface Lyt-1 than with Thy-1 were conslstenfly detected The proportions of T and B cells were reversely related An alteration in the number of cells bearing each antigen of 8% or more was consldered slgnificant (see Methods) The overall proportion of cells stained by each reagent was slgnificantly modified by treatment with only three of the compounds HCA, cyclophosphamide, or Ara-C (Figure 5) Treatment with cyclophosphamide greatly decreased the proportion of B cells, HCA had a slmilar but less pronounced effect Ara-C caused a modest increase m the proportion of T cells, but did not significantly decrease B cells Most of the drugs produced only minor shifts in the lymphocyte subpopulations defined by Lyt antigens, but two drugs, cyclophosphamlde and ABPP, caused changes which may be related to their effects on the immune response Cyclophosphamlde caused a very marked

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80

I

100

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I

I

40

I

i

60

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60

I

i

40

100

I

I

0

80

I

I

2O

[~[ Percent Lyt-l"2 + Cells

[ ] Percent Lyt-l+2 + Cells

I

0

I

i

100

20

!

80

• Percent Lyt-l+2" Cells

Spleen

I

40

!

I

I

60

60

40

Spleen

20

I

I

80

0

I

@

'11

100

Figure 4 The percent of cells in the thymus and spleen beanng Lyt-1, Lyt-2, or both antigens, in drug-treated mice Cells were stained wlth both fluorescem-labeled anti-Lyt-1 and rhodamzne-labeled antl-Lyt-2, and the percent Lyt-1 +2-, Lyt-1 +2 + and Lyt-1-2 + cells was determined using a microscope Data from one expenment are shown, 3 - 4 replicate samples (thymus) and 5 - 7 replicate samples (spleen) of 100 cells each were scored by two independent workers Values between the 100 cell samples did not vary more than 4% A second exper/ment gave nearly identical results

ABPP

15(S)-Me-PGE 1

Ara-C

Cyclophosphamide

Corticosterone

HCA

Saline

20

0

Thymus

O~

500

2 1,2,3

1,2,3

1,2,3

1

I

60

/

~

I

80

~

I

40

.

~

I

20

1

~ ' ~ ~ / ~

~ ~ ' / / ~

~

I

60

0

I

I

100

[ ] Percent Cells Ig Positive

I

80

I

100

~

~

~

~

~

~

I

40

80

I

I

20

60

I

I

40

40

I

I

60

20

I

I

80

0

I

I

100

Percent Cells Lyt-1 Positive

[ ] Percent Cells Lyt-2 Positive

100

I

I

0

•

Figure 5 Proportlon of cells in the spleens of drug-treated mzce that stazn wzth monoclonal antzbodzes speczfzc for Thy-1, Lyt-1, and Lyt-2, or with heteroant~serum agaznst mouse Ig The proportzon of cells wzth each surface antzgen was calculated by zntegratlng the area under the curves in the IF profzles Typical profzles are shown zn F~gure 6 The values shown are the mean of two experiments, except those for cortzcosterone (1 expenment) The results from the two expenments for all treatment groups agreed w~th~n a few percent Values for sahne controls Thy-1, 25 and 33%, Lyt-1, 35 and 41%, Lyt-2, 14 and 16%, and IgG, 54 and 62%

ABPP

15(S)-Me-PGE 1

50

250

Cyclophosphamide

Ara-C

125

Corticosterone

2

2

125

HCA

~

I

I

1,2,3

20

0

(mg/kg/day) (day)

Percent Cells Thy-1 Positive

•

Time

Dose

Saline

Drug

Drug Effects on Lymphocytes and Serum Corhcosterone

147

increased m the proporhon of splenocytes beanng Lyt-2 (Figure 5) The immunostlmulant ABPP on the other hand, reduced the number of cells beanng Lyt-2 These changes were reflected in both the Lyt-l+2 + and the L y t - l - 2 ÷ subpopulatlons (Figure 4) Figure 6 shows the log IF profiles of spleen cells from mace treated wlth cyclophosphamade or ABPP, each compared to saline-treated controls Because spleen cells were far more heterogeneous than thymocytes m the quanhty of antigens they bear, we collected the flow cytometry data using a Iogarithmlc, rather than a linear, amphfier of the fluorescence mtensaty The profiles of spleen cells from mlce treated wlth the other drugs were very s~milar to the controls In each profile, the left peak represents unstained and the nght peak stained cells Drug treatment produced only a few minor alterations m the peak poslhons (representing the denslty of the antigens on the resldual spleen cells)' ABPP reduced the density of Thy-1 and Lyt-1 on posltlve cells, and HCA reduced the denslty of Thy-1 (m some expenments) and Lyt-1 (not shown), cyclophosphamide reduced Lyt-1 density and increased Lyt-2 Surface Ig was dlmmlshed by cyclophosphamxde or ABPP We also wsually scored the Lyt-l+2 + spleen cells for denslty of anhgen expression in two experiments (Figure 7) The spleens of cyclophosphamide-treated m~ce had more dim Lyt-1/bright Lyt-2 cells while bnght Lyt-1/dim Lyt-2 cells were almost completely absent HCA produced similar shafts ABPP, on the other hand, reduced the populatlon of bnght Lyt-1/bnght Lyt-2 cells and increased the dlm Lyt-1/dim Lyt-2 cells

Corticosterone Production All of the drugs reduced the number of cells m both thymus and spleen All except the prostaglandin depleted the thymus of cells with a large quantity of surface Thy-1 and little Lyt-1 Since these drugs, whlch have a vanety of Lmmunomodulatory effects, all produced changes m thymocyte subpopulahons slmilar to those caused by hydrocortisone, we measured corhcosterone an the serum of drug-treated mlce, to determine whether the changes might slmply be mediated by drug-induced comcosteroid productlon ABPP or 15(S)-methyl PGEI caused a sagniflcant elevahon m the serum cortlcosterone measured three hours after a single treatment, but cyclophosphamide and Ara-C did not (Table 3) The concentrahon of co~costeroid an the blood 3 hours after rejection of ABPP was comparable to that produced by 125 mg/kg of exogenous cortlcosterone. Serum cortisol was also measured after drug treatment except in mlce treated wlth HCA, these levels dad not nse above 2 5/~g/dl F~gure 8 shows the serum corhcosterone concentrations at vanous t~mes after treatment wlth saline, corticosterone, 15(S)-methyl PGE I or ABPP The elevahon caused by ABPP was greater and more prolonged than that caused by the prostaglandm We also measured corhcosterone m serum from m~ce treated for three consecuhve days with Ara-C or ABPP (Table 4), since that dosing reglmen was used m the flow cytometry expenments The thlrd dose of Ara-C dld cause a modest increase in plasma corhcosterone, whereas the first dose did not In contrast, the first dose of ABPP ralsed comcosterone levels much more than the third To determine doses of HCA and cortlcosterone necessary to cause thymlc deplehon, mlce were rejected once mtrapentoneally w~th each drug at 125 mg/kg or at 12 5 mg/kg Only the hghest dose of co~costerone or HCA produced detectable comcostero~d levels m the serum Deplehon of thymic cells was observed 48 hrs after treatment wlth 125 or 12 5 mg/kg HCA, but corhcosterone was only effective at the highest dose (Table 5) To determine if the polyclonal activahon of B cells observed after treatment of mlce with ABPP might be due to induchon of corhcosterone, unimmumzed mlce were treated with HCA, comcosterone, or ABPP, and spontaneous PFC were measured m the spleen (Table 6) Treatment wlth ABPP increased the splemc PFC 9-fold, but nelther comcosteroid dld so

Th'r-1

Lyt-1

(n o

(J 14,..

o .Q

Lyt-2

E Z 4) .m 4.,*

w 4)

n

i

IgG

"'4

Log Fluorescence

Intensity

Figure 6 Effects of zmmunomodulatory drugs on the IF profiles of spleen cells Spleen cells were stained directly with fluorescezn-labeled monoclonal antzbodzes agaznst Thy-1 or Lyt-1 (I 20) or L yt-2 (1 100) or with heteroantzserum against m o u s e Ig (I. 100) The IF profiles were constructed using a Ioganthmic converter The dotted hnes zn all panels are IF profzles for sahne-treated mice T h e solid lines in the left panels represent ABPP-treated mzce while those on the nght represent cyclophosphamide-treated mzce In each profile, the peak on the left represents unstained cells IF profiles for HCA, cortzcosterone, Ara-C and 15(S)-methyl PGE I w e r e simzlar to controls

148

I

20

B

I II i m

EE

I

0 I

20

m BB El BB

I

0

I

20

Dim Lyt-1/ Dim Lyt-2

Figure 7 Denszty of Lyt-J and Lyt-2 antzgens on Lyt-1 +2+ spleen cells Spleen cells stazned szmultaneously with both fluorescezn labeled antz-Lyt-1 and rhodamzne-labeled anti-Lyt-2 were scored vzsually for the brightness of red and green staznzng: 100 cells posltwely stained for Lyt-2 were scored Data from one experiment are presented Very szmzlar results were obtazned zn a second expenment

~

I

0

15(S)'Me'PGE1

i

80

m.. m ~ B

i

60

.c. ~ Cort,coste.o.~ c.o,o.hos.ha~,.e .ra-C ~

i

40

EE

|

i

Dim Lyt-1/ Bright Lyt-1/ Bright Lyt-2 Dim Lyt-2

S...e

20

0

Bright Lyt-l/Bright Lyt-2

Table 3

S e r u m cort~costerone ° concentrations in mzce three hours after treatment

Treatment b

Cortlcosterone l.~g/dlc

None Sahne HCA Cort]costerone Cyclophosphamlde Ara-C 15(S)-methyl PGE I ABPP

14 8 66 118 25 19 63 97

± 4 ± 2 _+ 15 d ± 61 d ± 16 + 9 ± 3d ± 12 d

a The cort]costerone RIA measures both co~costerone and HCA The c o . s o l level m mice treated with HCA (measured us]ng a d]fferent RIA method) was equal to that detected by the cort]costerone assay, but m mlce treated with all other compounds ]t was ~ 2 5 Fg/dl bBlood samples were obtained three hours after a single treatment wlth the drugs, us]ng the doses and routes shown m Table 1 Samples from some untreated controls were taken before and some after bleeding the treated m~ce CThe values reported are the means +_ standard dewahon for 5 ]nd,v, dual mlce per group (each done in duphcate) dSlgnlflcantly dlfferent from sahne (p ~ 0 05) by the Kruskal-Walhs test

Figure 8

S e r u m cortrcosterone concentration in mlce as a functzon of time after treatment (the m e a n o f 5 mzce p e r group, +_ standard clevlat~on) T h e asterisks zndicate values slgmfzcantly dlfferent (p ~ 0 0 5 ) f r o m the saline control Untreated mlce had serum cortzcosterone concentrat)ons o f ~ 13 ~g/dl

240

-

220 200 180 •o

160

:=L ""

140

o c o t/J

@

120

o o

100

o

80

Corticosterone

0

ABPP

6O 40

i

20

~(~ S a l i n e I 2

I 3

I 4 Hours

150

15(S)-Me-PGE

I 5

I 6

1

Table4

Comparisonoftheeffectsofthefzrstandthirddailytreatmentwithdrugsontheserum corticosterone ° concentration of m~ce Serum cortzcosterone concentration ( I.ug/dl) Three Treatments

Treatment b

O n e Treatment

Saline Ara-C ABPP

8 +- 2 19+_ 9 97 -+ 12 c

9-+ 4 43+- 7 c 20 +- 10 c

a C o ~ c o s t e r o n e concentrations were measured on serum obtained three hours after (the final) treatment bDoses and routes of admmlstratlorl are shown m Table 1 CS1gnlficantly dlfferent (p ~ 0 05) from sahne by the Kruskal-Wallis test At 125 mg/kg, corticosterone produced a serum concentration of 118 _ 57, and HCA, 66 - 15 p~g/dl, respectively

Table 5

Effect of H C A or cortzcosterone treatment on thymus cell number" Dose (mg/kg)

Treatment Sahne HCA HCA Co~costerone Corticosterone

-12 125 12 125

Mean Number of Viable Cells~Thymus hn mllhons)

% Viable b

126 28 3 125 19

97 95 79 98 92

5 0 5 0

aThe results reported are those from a single e x p e n m e n t but are representatlve of several e x p e n m e n t s There were five mlce per group The anlmals were treated 48 hrs prior to harveslang the cells bViablhty was assessed by excluslon of 0 05% trypan blue

Table 6

Spontaneous PFC a zn the spleen of unimmumzed, drug-treated mice

(mg/kg/day)

(days)

Nucleated Spleen Cells (m mdhons)

-50 50 250

1,2,3 i 1 1,2,3

98 72 97 117

Dose

Drug b Sahne HCA Co~costerone ABPP

Tzmec

PFC/Spleen cl 100 60 70 900

--_ 40 + 20 -+ 20 - 160 e

aMeasured uslng SRBC as targets bAll drugs were admmlstered i p CRelalave to assay on day 4 ~The values s h o w n are the m e a n s (+ standard dewa~ons) for 6 - 9 mlce per group (each m o u s e assayed in quadruphcate) eS1gmflcantly ddferent from saline (p ~ 0 05) by the Kruskal-Walhs test 151

152

P E Fast et al

DISCUSSION Enumeration of lymphocytes bearing various surface antigens may provide important clues to the mechanisms of action of immunomodulatory drugs and clarify the interpretation of functional studies This study used immunofluorescence staining coupled wlth microscoplc and flow cytometric analysis to identify the lymphocyte subpopulations in mice treated with immunomodulatory drugs and to relate drug-induced changes to immunological function Flow cytometry provided information on the antigen density Microscopic examination of cells stained simultaneously with rhodamme-labeled anti-Lyt-2 and fluorescein-labeled anti-Lyt-1 permltted enumeration of the Lyt-1+2 -, Lyt-l+2 +, and Lyt-l-2 ÷ subclasses. HCA, cyclophosphamide, Ara-C, and ABPP, despite their different mechanisms of actlon, caused rather similar alterations m the thymus, cells bearing high Thy-1 and low Lyt-1 were selectively killed or emigrated from the thymus The density of Lyt-2 antigen on the surviving cells was quite similar to that m the normal thymus, although the proportion of cells bearing Lyt-2 was decreased. The reciprocal shlfts m density of Lyt-1 and Thy-1 caused by HCA, with relatively httle alteration in the distribution of Lyt-2, have been described by others (Fathman et al, 1975, Mathieson et al, 1979; Ledbetter et al, 1980, Scollay and Weissman, 1980), but lymphocyte subpopulations have not been enumerated in animals treated with other immunomodulatory drugs except diethylstilbestrol (Kalland, 1980) All the drugs we studied decreased the Lyt-l-2 + subpopulation m the thymus and they depleted the Lyt-l+2 + population to varying degrees The most likely interpretatlon of the effects of HCA, Ara-C, and cyclophosphamide on the thymus is that the drugs destroy most of the original cells The completeness of thym~c atrophy seems to be roughly correlated with the size of the remaining cells. Apparently, the mare target of all these drugs--regardless of their mechanism of actlon--ls thymocytes bearing Lyt-2. These data substantiate the conclusion of earlier workers that cyclophosphamide and HCA destroy for the most part the same thymocyte subpopulations (Dumont and Barrols, 1975) We have previously shown that the drug-resistant thymus cells have a higher cholesterol/phospholipid ratio than the predominant thymocytes m untreated ammals (Merntt et al, 1982) Apparently, maturatlon and differentiation of thymus cells are accompanied by alterations in the hpld membrane composition as well as antigenlc expresslon Alternatively, changes in the hp~d composition during maturation m~ght affect antigenic expresslon The similarity of the effects of Ara-C, cyclophosphamide, and ABPP on the thymus to those of HCA, ralsed the possibility that the former compounds cause stress-induced release of corticosteroids Measurement of serum co~costerone levels after administration of these compounds showed a modest elevation caused by the prostaglandin and ABPP but not by Ara-C or cyclophosphamlde The study of co~costerone productlon as a result of drug treatment is an essential feature of the evaluation of any new drug that alters ~mmune funclaon m rodents, since rodents are far more susceptible to steroid-mediated immune depletlon than man Exogenously admimstered corticosterone, the principal natural corticostero~d of m~ce (Ganong, 1979), had far less effect than HCA on the number, slze and antlgemc markers of the thymocytes. Of the immunomodulatory drugs tested, ABPP was the only one whch produced serum levels of corticosterone comparable to those produced by an rejection of 125 mg/kg of corticosterone itself (The pharmacokinetics of endogenously produced and exogenously administered corticosterone are quite different, so this comparison ~s not precise ) The quantity of corticosterone reduced by ABPP might be sufficient to cause thym~c depletion and a shift in antigemc subpopulations, but it is insufficlent to inhlbit the GVH reactlon (125 mg/kg exogenous corticosterone did not suppress the splenomegaly) The most stnking effect of ABPP, polyclonal activation of B cells to produce immunoglobulin, cannot be due to co~costerold production, since nezther corticosteroid mimlcs this effect Much more corticosterone ~s secreted in response

Drug Effects on Lymphocytes and Serum Corticosterone

153

to the first, than the third dose of ABPP T h s parallels the interferon response; interferon is produced slx hours after the first dose of ABPP but mice are hyporeactive to the second or third dose (Stringfellow et a l , 1980) The connection between these two phenomena Is not clear 15(S)-methyl-PGE I also caused a modest elevation of serum co~costerone, but too little to explain its effects on the GVH reaction The data in thls paper show that cyclophosphamide does not act via corticosterone production, nor does Ara-C. Stimulation of corticosterone production however, may be the means by which ABPP causes depletion of thymocytes In the spleen, unlike the thymus, the p r o p o ~ o n of dlfferent cell subpopulations was changed by drug treatment in only a few instances HCA and cyclophosphamlde both depleted the spleen of B cells with a corresponding increase in the proportion of T cells The somewhat surpnsmg excess of cells with measurable Lyt~l over those wlth measurable Thy-1 m all treatment groups, also observed by others (Ledbetter, et al., 1980; Loveland et al., 1981), is probably explained by the finding that B cells may express the Lyt-1 antigen (Lanler et al, 1981) At high doses, cyclophosphamide is a powerful immunosuppresslve drug, at lower doses, its actions are more complex (Askenase et al, 1975, Hardt et al, 1981) It inhibited antibody formation in our study, reduced spleen weight in GVH and non-GVH rmce, and depleted B cells more than T cells (see Shand, 1979, for a revlew of previous work) B cell depletlon by cyclophosphamlde has been observed previously (Turk and Poulter, 1972) The reduction we observed in B cells probably was insufficient to explain the total inhibition of antlbody formation It seems hkely, however, that the proportional increase of Lyt- 1-2 + and Lyt- 1+2 + spleen cells of cyclophospham~de-treated mice is responsible for at least some of the ~mmunomodulatory actions of the drug Cyclophosphamide treatment of ummmunized mice induces suppressor cells charactenzed as non-T cells (Mclntosh et al, 1979) or as T cells (Braciale and Pansh, 1980) Merluzm et al (1980) have shown that cyclophosphamlde depletes a population of helper cells required for the generation of T killer cells. They characterized the helpers as Lyt 1+2- cells because they were not killed by anti-Lyt-2 antibodies and complement. Their results, suggesting that cyclophosphamlde depletes the spleen of Lyt-i+2- cells, seem to conflict wlth our observation that cyclophosphamide treatment does not alter this population Our visual scoring of Lyt-1 a n d Lyt-2 staining intensity s h o w e d , however, that the Lyt-I ÷ 2 + subpopula~on is itself heterogeneous, containing cells wlth large and small quantltles of each antlgen In the spleens of cyclophosphamlde-treated mlce, cells wlth a high density of Lyt-1 antigen but a small amount of the Lyt-2 antigen are reduced by about 80%. We propose that the "Lyt-l+2 - ' ' helper cells described by Merluzm and colleagues may be Lyt-l+2 ÷ cells expressing such low levels of the Lyt-2 antigen that they are not killed by antlbody against Lyt-2 Ledbetter et al. (1980) found that Lyt-l+2 + cells are difficult to kill with anti-Lyt-1 antlbody and complement Unlike cyclophosphamide, ABPP stimulates antibody responses Treatment wlth ABPP seems to hft a natural suppresslon of spontaneous antibody formation and facilitates antibody response to antigen (Fast and Stnngfellow, 1980) It reduces the proportion of both Lyt-l*2 + and Lyt-2 + cells m the spleen, thus presumably reducing suppressor clrcults and increasing help (Cantor and Gershon, 1979) The suppresslve action of ABPP on the GVH reaction is more difficult to explain, but its activlty is found late in the reaction, when cells bearing Lyt-2 predominate (Fast-~nd Hatfield, unpublished data), so the reduction in Lyt-2 + cell populations may be relevant It seems hkely that in the cases of cyclophosphamide, diethylstilbestrol (Kalland, 1980) and ABPP, analysis of the lymphocyte subpopulations m the spleen g~ves a significant mslght into the mechanism of action of the drug In other cases, such as Ara-C, a drug which acts by altering metabolic activities of dlwding cells (Boggs et al, 1973), or 15(S)-methyl PGEI, which appears to act pnmanly on the effector phase of immuno-mflammatory reactions (Kunkel et al 1980) there is no slgnificant change in the proportion of suppressor, helper, amplifier, or feedback

154

P E Fast et al

suppressor cells in the penphery Enumeration of lymphocyte subclasses using Lyt and Qal antigen (Cantor and Gershon, 1979) or their human equivalents will be an important tool in unraveling the mechanism of action of drugs which modulate rather than simply inhibit immune responses

We thank E L Sun and K A Anms for help in prepanng cell suspensions, K R Lamborn for statlsbcal advice, and S K Moyerfor editorial assistance

REFERENCES Askenase PW, Hayden BJ, Gershon RK (1975) Augmentation of delayed-type hypersensitivity by doses of cyclophosphamide which do not affect antibody responses J Exp Med 141 697 Besedovsky HO, del Ray A, Sorkin E (1981) Lymphokine-containmg supernatants from Con A stimulated cells increase corticosterone blood levels J Immunol 126:385 Besedovsky H, Sorkin E (1977) Network of immune-neuroendocnne interactions Chn Exp Immunol 27' 1 Boggs SS, Boggs DR, Nell GL, Sartiano G (1973) Cycling characteristics of endogenous spleen colony-forming cells as measured with cytosine arabmoside and methotrexate J Lab Clin Med 82.727 Braciale VL, Parish CR (1980) Inhibltion of m vitro antibody synthesis by cyclophosphamldeinduced suppressor cells Cell Immunol 51' 1 Calkins CE, Stanton TA, Stutman O (1980) Cellular requlrements for the in vitro reduction of specific suppression of antibody response by immune spleen cells Eur J Irnmunol 10 449 Cantor H, Asofsky R (1970) Synergy among lymphold cells mediating the graft-versus-host response II Synergy in graft-versus-host reactions produced in BALB/c mice by lymphoid cells of differing anatomic ongln J Exp Med 131 235 Cantor H, Boyse EA (1977) Lymphocytes as models for the study of mammahan cellular differentiation Imrnunol Rev 33-105 Cantor H, Gershon RK (1979) Immunological circuits: cellular composition Fed Proc 38 2058 Claman HN (1972) Corticosteroids and lymphoid cells N Engl J Med 287 388 Cohen J J, Fischbach M, Claman HN (1970) Hydrocortisone resistance of graft vs host activity in mouse thymus, spleen, and bone marrow J Immunol 105:1146 Dumont F, Barrols R (1975) Effect of treatment with cyclophosphamide on the electrophoretic mobility and mltogen responsiveness of mouse thymus cells Biomedicine 23 391 Fast PE and Stringfellow DA (1980) Immune modulation by two antiviral isocytosines wth different abilities to induce interferon In Current Chemotherapy and Infectious Dzsease Eds, JD Nelson and C Grassi Washington DC The American Society for Microbiology, pp 1396-1398 Fathman CG, Small M, Herzenberg LA, Weissman IL (1975) Thymus cell maturation II Differentiation of three "mature" subclasses in vivo Cell Imrnunol 15 109 Ganong WF (1979) Rewew of Medical Physiology Los Altos, CA Lange Press. p 283 Hardt C, R611inghoff M, Pfizenmaier K, Mosmann H, Wagner H (1981) Lyt23+ cyclophosphamlde-sensltive T cells regulate the activaty of an interleukin 2 inhibitor In vivo J Exp Med 154'262 Herzenberg LA, Herzenberg LA (1978) Analysis and separation using the fluorescence activated cell sorter (FACS) In Handbook of Expenmental Immunology, vol 2, 3rd edition Ed. DM Weir London Blackwell Scientific Publications, pp 22 1 - 2 2 21 Howard JG, Woodruff MFA (1961) Effect of the graft-vs-host reaction on the immunological responsiveness of the mouse Proc R Soc Lond (B) 154 532 Jeme NK, Nordin AA, Henry C (1963) The agar plaque technique for recognizing antibody

Drug Effects on Lymphocytes and Serum Cortlcosterone

155

producing cells. In Cell Bound Antlbodies Eds, B Amos and H Koprowski Philadelphla W~star Institute Press, p 109 Kalland T, (1980) Decreased and dispropo~onate T-cell populatlon m adult mlce after neonatal exposure to dlethylstilbestrol Cell lmmunol 51'55 Kunkel SL, Thrall RS, Kunkel RG, McCormlck JR, Ward PA, Zuner RB (1979) SuppressLon of immune complex vascuhtls m rats by prostaglandln d Chn Invest 64 1525 Lanier LL, Warner NL, Ledbetter JA, Herzenberg LA (1981) Expresslon of Lyt-1 antigen on certain murlne B cell lymphomas J Exp Med 153.998 Ledbetter JA, Rouse RV, Mlcklem HS, Herzenberg LA (1980) T-cell subsets defined by expresslon of Lyt-l,2,3 and Thy-1 antlgens two-parameter immunofluorescence and cytotoxicxtyanalysls with monoclonal antibodles modifies current views d Exp Med 152 280 Loveland BE, Hogarth PM, Ceredig R, McKenzie IFC (1981) Cells mediating graft rejection in the mouse I Lyt-1 cells mediate skin graft rejection d Exp Meal 153.1044 Mathleson BJ, Sharrow SO, Campbell PS, Asofsky R (1979) An Lyt differentlated thymocyte subpopulataon detected by flow mlcrofluorometry Nature 277 478 Mclntosh KR, Segre M, Segre D (1979) InhlblUon of the humoral response by spleen cells from cyclophospham~de-treated mlce Immunopharmacol 1 165 Merluzzl VJ, Faanes RB, Choi YS (1980) Recovery of the capaclty for cyotoxlc T cell generatlon m cyclophosphamide-treated mlce by the addihon of Lyt-l+2-helper cells Int d Iramunopharmacol 2 341 Merritt MV, Llcht NJ, Hatfield CA, Fast PE (1982) Membrane flmdlty and relatlve cholesterol content of thymus and spleen cells from mlce treated wlth immunomodulatory drugs Immunopharmaco! (to appear) Mlshell RI, Dutton RW (1967) Immumzatxon of dlssocaated spleen cell cultures from normal mlce J Exp Meal 126 423 Raff MC (1969) Theta Isoantlgen as a marker of thymus-derived lymphocytes in mlce Nature 224 378 Remherz EL, Kung PC, Goldstem G, Schlossman SF (1979) Separatlon of functlonal subsets of human T cells by a monoclonal antlbody Proc Nail Acad Sc~ USA 76 4061 R11ey V (1981) Psychoneuroendocrine influences on ammunocompetence and neoplasia $clence 212 1100 Rotman B, Papermaster BW (1966) Membrane propertLes of hwng mammahan cells as studied by enzymatlc hydrolysls of fluorogemc esters Proc Natl Acad Sc~ USA 55 134 Scollay R, Kochen M, Butcher E, Welssman I (1978) Lyt markers on thymus cell migrants Nature 276 79 Scollay R, Welssman IL (1980) T cell maturataon thymocyte and thymus mlgrant subpopulatlons defined wlth monoclonal antibodies to the antlgens Lyt-1, Lyt-2, and ThB d Immunol 124 2841 Shand FL (1979) The Immunopharmacology of cyclophosphamlde Int J Irnmunopharmacol 1 165 Shortman K, Wflhams N, Adams P (1972) The separatlon of dlfferent cell classes from lymphold organs V Simple procedures for the removal of cell debris, damaged cells, and erythrold cells from lymphoid cell suspenslons d Immunol Methods 1 273 Stnngfellow DA, Vanderberg HC, Weed SD (1980) Interferon reduction by 5-halo-6-phenyl pynmldmones J Interferon Res 1 1 Turk JL, Poulter LW (1972) Selectlve depletlon of lymphold tlssue by cyclophosphamlde Chn Exp Immunol 10 285