Membrane fluidity and cholesterol in thymus and spleen cells from mice treated with immunomodulatory drugs

Membrane Fluidity and Cholesterol in Thymus and Spleen Cells from Mice Treated with Immunomodulatory Drugs* Margaret V. Merritt,* Norman J. Licht,* Cheryl A. Hatfield,** and Patricia E. Fast** Abstract: We have used spin labeling, fluorescence polarization, and chemical analysis to characterize membrane properties of thymocytes from mice treated with immunomodulatory drugs. The number of thymocytes was reduced 90--95% by treatment of 6--9 week old mice with hydrocortisone acetate (HCA) or methylprednisolone (both 125 mg/kg) or with cyclophosphamide (250 mg/kg). Electron spin resonance (esr) examination of thymocytes labeled with 5-nitroxyl stearic acid indicated that the membranes of cells remaining after treatment with any of these drugs were more rigid than those from saline-treated controls. The total cholesterol/ phospholipid (C/PL) molar ratio of the HCA-resistant thymocytes was twice that of the control mice. Treatment of mice with other immunomodulatory drugs, cyclophosphamide, cytosine arabinoside (Ara-C), 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP) and 15(S)methyl prostaglandin E t (15(S)-methyl PGE I), also altered the C/PL ratio in thymocytes and, in some cases, in spleen cells. Fluorescence polarization measurements of thymocytes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) did not reveal the differences between cells from HCA-and saline-treated mice that were detected by spin labeling and chemical analysis. Our results indicate that the greater rigidity detected by spin labeling of hydrocortisone-re,~stant thymocytes may be due. at least in part, to greater membrane cholesterol content. Of the methods employed, chemical analysis was the most sensitive in revealing drug-induced alterations in thymocyte populations.

Key Words: Hydrocortisone acetate; Methylprednisolone; Cyclophosphamide; Cytosine arabinoside (Ara-C); 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone (ABPP); 15(S)-methyl prostaglandin E I (15(S)-methyl PGEI) membrane fluidity; Spin labeling; Fluorescence polarization; 1,6-diphenyl-l,3,5hexatriene (DPH); 5-nitroxyl stearic acid; Lymphocytes; Cholesterol; Phospholipid.

"For a preliminary report, see Merritt et al., 1980. A portion of the present work was submitted by N.J. Licht to Kalamazoo College as a Senior Thesis. Received July 26, 1981; revised March 24, 1982. From *Physical and Analytical Chemistry Research and "*Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, Michigan 49001. Address requests for reprints to Dr. Margaret V. Merritt, Deparlment of Chemistry, Wellesley College, Wellesley MA 02181. Norman J. Licht and Patricia E. Fast are currently at the College of Human Medicine, Michigan State University, East Lansing, MI 48824. © Elsevier Science Publishing Co., Inc., 1982 52 Vanderbflt Ave., New York, N.Y. Immunopharmacology 5. 49-64 (1982)

49 0162-3109/82/0504916502.75

50

MV. Merritt et al.

INTRODUCTION The purpose of this study was to determine the effects of immunomodulatory drug treatment on lymphoid cell characteristics and to compare spin labeling, fluorescence polarization, and direct chemical analysis as methods for monitoring alterations. We included a corticosteroid in the study because of the extensive information available on the pharmacological effects of this class of drugs upon lymphoid tissues and cells (Claman, 1972). Corticosteroid treatment depletes human lymphoid tissues little if at all, but in the mouse, thymus weight drops rapidly and markedly following systemic administration of these drugs. Steroid treatment profoundly depletes the cortical cells (cortisone-sensitive, CS) and leaves the medullary thymocytes (cortisone-resistant, CR) intact (Ishidate and Metcalf, 1963; Dougherty et al., 1964). CR thymocytes, which constitute about 5% of the adult mouse thymus, are immunologically competent: they can cause graft vs host (GvH) reactions (Blomgren and Anderson, 1969), function as helper cells in the production of humoral antibodies against certain antigens (Blomgren and Anderson, 1971) and kill antibody-sensitized target cells (Lamon et al.. 1978). Steroid treatment also depletes spleen cells. The CR spleen cells are mature T-cells; they cause GvH reactions (Cohen et al., 1970), suppress both antitumor immune response (Schecter and Feldman, 1977) and autoimmunity (Morton et al., 1976), proliferate in response to mitogens (Fathman et al., 1975) and contain the precursors of cytotoxic T lymphocytes (Babu and Sabbadini, 1977). The physical characteristics of CR and CS murine thymocytes differ in several respects. Dumont has shown that CR thymocytes have a greater surface charge than the three electrophoretically distinguishable subpopulations of CS thymocytes (Dumont, 1978). CR thymocytes have the lowest buoyant density of the thymocyte subpopulations (Droege el al., 1974) and appear to have a higher cholesterol/phospholipid ratio than CS thymocytes (Kigoshi, 1978). CR medullary thymocytes express less Thy-I antigen (Fathman et al., 1975; Zeiler et al., 1974) and more Lyt-1 (Ledbetter et al., 1980; Mathieson, et al., 1979) than the small CS immunoincompetent cells of the thymus cortex. Previous studies have indicated that CR thymocytes are the mature cells of the thymus (Blomgren and Anderson, 1971; Fathman et al.. 1975; Zeiler et al., 1974). Fathman et al. (1975) have shown that the CR thymocytes are indistinguishable from a subpopulation of medium-sized cells which make up less than 10% of all thymus cells, but information is incomplete regarding the relationship of antigenic markers to electrophoretic mobility and the physical and chemical properties of CR and CS spleen cells. Very little work has been done using other immunomodulatory drugs. We report the results of spin labeling, chemical analysis, and fluorescence polarization studies to characterize further the physical properties of both thymocyte and spleen cell membranes from mice treated with HCA and other immunoregulatory drugs with diverse mechanisms of action.

MATERIALS AND METHODS Mice and Drug Treatment All animals used in these studies were 6 - 9 weeks old unless otherwise noted. The CBA and (C57BI/6xCBA) F I mice, referred to as B6CBF I, were females obtained from Cumberland View Farms, Clinton, TIN or Jackson Laboratories, Bar Harbor, ME. Male AKR and C57BI/6

Abbreviations: ABPP: 2-amino-5-bromo-6-phenyl-4-(3H)-pyrimidinone; Ara-C: cytosine arabinoside; C/PL: cholesterol/phospholipid; CR: cortlsone-resistant; CS: cortisone-sensitive; DPH: i ,6-cLiphenyl-l,3,5-hexatriene; esr: electron spin resonance; GvH: graft vs. host; HBSS: Hanks' balanced salt solulion; HCA: hydrocortisone acetate; i.p.: intraperitoneal; 5-NS: 5-nitroxide stearic acid; P: fluorescence polarizalion; PBS: phosphate buffered saline; 15(S)-methyl PGEI: 15(S)-methyl prostaglandin E l.

Membrane Fluidityof Murine Lymphocytes

51

mice were purchased from Jackson Laboratories. The six-day old CBA mice used in these experiments were of both sexes. Drug suspensions or solutions were prepared for each experiment by mixing saline or 0.1% Tween 80 in saline with weighed samples of the drug. The chemical structures of the compounds are shown in Figure 1. All of the drugs except cyclophosphamide (Fairfield Chemical, Blythewood, SC or Mead Johnson, Evansville, IN) came from the Upjohn Company. Animals treated with HCA or with methylprednisolone were given a single intraperitoneal (i.p.) injection of 125 mg/kg 2 days before thymus and spleen cells were examined. Those treated with cyclophosphamide received 250 mg/kg, i.p., 3 days before the thymus and spleen were removed. Mice were injected i.p. daily for 3 days with 50 mg/kg/day Ara-C or 500 mg/kg/day ABPP. 15(S)-methyl PGE 1 was dissolved in 95% ethanol and diluted with 11.5 volumes of saline just before oral administration; mice received 2 mg/kg/day for 3 consecutive days and were killed on the fourth day. Animals treated with medroxyprogesterone acetate were injected i.p. with 100/mg/kg 2 days before they were killed. These doses and routes of administration were selected to be immunoregu]atory and nontoxic. Control animals in each experiment were injected with equal volumes of saline or 0. 1% Tween 80 in saline. Animals were killed by CO2 inhalation and the thymuses and spleens were

Figure I

Structures of drugs used in this study. CHz-O-C-CH 3 I IJ

CH20H I

C=O0

C=O

O~'V,~'v ~

O ~ V ~ , CH3

HCA

METHYLPREDN I SOLONE

NH2 0 ml

OH

.CH2(CH2)5CO2H

H3C

OH

0~

N~N N '~

Br~)U.NH~N ~NH

H

HOCH20.1

OH

15(S)-METHYL

PGEI

H

C.o P

CHzCHzCI

0/ \N / k CH2CH2CI CYCLOPHOSPHAMIDE

ABPP

ARA-C

~

CH 3 I C=0 -O-C-CH II

0

MEDROXYPROGESTERONE ACETATE

3

52

M.V. Merritt et al.

removed and placed immediately in ice cold Hanks' Balanced Salt Solution (HBSS) buffered with 0.01 M HEPES (GIBCO, Grand Island, NY). This medium was used unless otherwise indicated. For each spin labeling and fluorescence polarization experiment. 30 mice received each drug and 5 mice served as saline-treated controls. Lipid analyses were performed on extracts of the pooled thymuses or spleens from 7 to 13 animals per group. Cell Preparation Fascia and connective tissue were removed, and single cell suspensions in buffered HBSS were prepared by forcing the freshly removed tissue through an 80 gauge stainless steel screen. Erythrocytes were removed from spleen cell suspensions by lysis with ammonium chloride (Shortman et al., 1972); the cells were suspended in an ammonium chloride solution (0.15 M), buffered at pH 7.2 with 0.02 M Tris, for about ten minutes at 20°C until microscopic examination showed that lysis was complete. The cells were diluted immediately with cold buffered HBSS and washed at least 3 times: the number of viable nucleated cells (viability assessed by exclusion of 0 0 5 % trypan blue) was counted using a hemacytometer. The minimum acceptable viability was 70%. although, in most experiments, 9 0 - 9 5 % of the thymus cells were viable.

Fluorescence

Polarization

Single cell suspensions were prepared from thymuses as described above: the final suspension was washed once and resuspended at 4 × 106 cells/ml in 0.001 M phosphate buffered saline, pH 7.2 (PBS). Aliquots of this suspension were mixed with equal volumes of a freshly prepared PBS solution of 1.0 x 1 0 6 M 1,6-diphenyl-1,3,5-hexatriene (DPH, Aldrich, Milwaukee, WI). The solution was prepared by rapidly injecting a 25 p,l aliquot of a tetrahydrofuran solution of DPH (I x i 0 3 M) into 25 ml of PBS. The cells were incubated for 10 minutes at 37°C, washed once in PBS and resuspended at a final concentration of 2 x 106/ml. Unlabeled control cells in PBS were prepared at the same concentration. Fluoresence measurements were made on a Perkin-Elmer (Norwalk, CT) MPF-2 spectrofluoremeter equipped with polarizing filters with excitation at 365 nm and emission of 450 nm; slit widths were 10 nm. The cell suspensions were kept at 37.0 _+ 0.I°C using a Haake recirculating temperature bath. The fluorescence polarization value, P, was calculated as follows (Chen and Bowman, 1965; Fuchs et al., 1975): p=

I~.~ - |v.h ' [h.~/lh.h Iv.v + l~,h • lh,v/lh.h

where I is the corrected fluorescence intensity and the subscript v indicates vertical orientation of the excitation and analyzer polarizers and h indicates horizontal orientation. The fluorescence intensity of the unlabeled cells was zero.

Spin Labeling An aliquot of an ethanol solution of 5-nitroxide stearic acid (5-NS, Syva Associates, Palo Alto, CA) was transferred to a glass Edenmeyer flask and the ethanol removed by evaporation with nitrogen. Approximately 4 x 108 cells were labeled at one time in a 50 ml Erlenmeyer flask; 10 -2 mg of 5-NS was used for each 107 cells. Cells at a concentration of 20 x 106/ml in buffered HBSS were added to the flask, followed by a sufficient volume of a 10% solution of bovine serum albumin (Reheis Chemical Co., Phoenix, AZ) to give a final concentration of 0.1% by weight. The flask was covered and incubated for 1 hr in an ice-water bath with gentle agitation (Dubnoff Metabolic Shaking Incubator). Then the total suspension was transferred to a clean Erlenmeyer flask. Aliquots for electron spin resonance (esr) examination were taken from this

Membrane Fluidity of Murine Lymphocytes

53

suspension, which was kept in the shaking ice-water bath for no more than 4 hours. The viability of labeled cells was no less than that of the original unlabeled cell preparation. A specially designed esr centrifuge tube was used for these studies. It consisted of a 5-ml disposable pipet tip (Rainin Instrument Co., Brighton, MA) with the tapered end closed with a Teflon plug; the volume in the conical tip was about 40/~I. For each esr sample, a 1.0-ml aliquot of the above cell suspension (2 × 107 cells) was transferred to the esr centrifuge tube and centrifuged at 4°C for 5 min at 450xg. About 0.95 ml of supematant was removed and the cells were resuspended in the remaining medium. This suspension was transferred with an Eppendorf pipet to an esr sample cell composed of Teflon tubing having an approximate length of 6 inches with a 1/32 inch internal diameter. The ends of the tubing were sealed with Red Wax (Cenco, Chicago, IL). The tubing was inserted into a 3-mm outer diameter quartz tube filled with silicone fluid (Dow Coming 200, Midland, MI). This assembly was then inserted into the cavity of the esr spectrometer.

Electron Spin Resonance Measurements All esr measurements were made on a Varian E-9 spectrometer using an E-232 dual sample cavity with a variable temperature insert (Varian Instruments, Palo Alto, CA). Temperature regulation was achieved with a Varian E-257 variable temperature controller. The sample temperature was continuously monitored with a Newport Digital Pyrometer (Model 267) via a copper-constantan thermocouple mounted in the silicone fluid of the esr sample tube just above the active region of the cavity. Less than a 1°C temperature gradient existed across the sample. All measurements were made at 36 -+ 2°C. The spectra were recorded using 100 KHz modulation with an amplitude of 2.5 gauss at a power of 50 roW. The signals were not saturated at this power. The time required for recording the esr spectrum (4 rain) was short relative to the time required for other parts of the experiment. The spectrometer was interfaced to a Nicolet 1180 computer for data storage and reduction. The values of 2Areax were determined from these stored data. In several experiments, the same values of 2Ar~, were measured independently by two workers who did not know the identity of the samples. The utility and applicability of 2Amax have been well established (Keith and Mehlhorn, 1972; Butterfield et al., 1974; Jost et al., 1971).

Extraction of Lipids Single cell suspensions were prepared as described above. The cells were resuspended in 1.0 ml PBS in a 15 ml glass centrifuge tube. The lipids were then extracted by a modification df the method of Bligh and Dyer (1959) as follows: a 3.75 m] aliquot of methanol/chloroform (2: 1) was added, and the tube was shaken for 40 minutes. The upper layer was transferred to a clean centrifuge tube. The residue was extracted with 4.75 ml of methanol/chloroform/water (2: 1:0.8) by shaking for 10 minutes. After centrifugation, the upper layer was combined with the first extract. Chloroform and water, 2.5 ml each, were added to the combined extracts. The lower chloroform layer was removed, mixed with an equal volume of toluene and the solvent removed with a rotary evaporator. The lipid residue was immediately dissolved in 10.0 ml of chloroform and stored in the cold. Aliquots of this chloroform solution were analyzed for phospholipid and cholesterol content.

Cholesterol Analysis Aliquots (0.5-2.0 ml) of the chloroform extract solution were transferred to clean test tubes and the solvent removed with a stream of nitrogen. The residue was dissolved in 200/~1 of isopropyl alcohol. To each tube was added 400/~1 of ferric chloride reagent (30 mg of FeC13/100 ml of

54

M.V. Merrill et al.

glacial acetic acid) followed by 400 p,l of concentrated sulfuric acid. The tubes were allowed to sit for 10 minutes before the absorbance was measured at 540 nm. The cholesterol content was determined from a standard curve prepared from samples of a stock solution of cholesterol (Applied Science, State College, PA) treated in an identical fashion. This procedure is essentially that of Zlatis et al. (1953).

Phospholipid Analysis The phospholipid content of the extract was determined as inorganic phosphate in digested samples following a modification of the method of Morrison (1964). Aliquots of 5 0 - 3 0 0 #,I of the chloroform lipid extract were pipeted into glass test tubes and the chloroform removed with a stream of nitrogen. A standard phosphate solution (0.2 mg/ml) was prepared from analytical reagent grade Na2HPO 4 (Mallinckrodt, St. Louis, MO). To an aliquot of the phosphate solution or dry sample of lipid was added 0.3 ml concentrated sulfuric acid and 50 p,l of 30% H20 2 (Mallinckrodt). The lest tubes were heated over a small Bunsen burner until the solution was clear and gas evolution had ceased. Four ml of water along with I00 p,l of 33% NazSO 3 was added with thorough mixing. Following addition of 1.0 ml of 2% (NH4)0MoTO24-4H20, 10 mg of solid ascorbic acid was added and the solution mixed thoroughly. The samples were heated in a boiling water bath for I0 minutes, cooled and extracted with a 3.0 ml of n-pentanol (Aldrich, Milwaukee, WI). The absorbance at 795 nm of the blue pentanol layer was measured. The phosphate in the lipid samples was determined from the standard curve derived from the adsorbance of inorganic phosphate samples.

RESULTS Treatment of mice with HCA, methylprednisolone, cyclophosphamide or Ara-C reduced the number of viable thymocytes recovered from each mouse by about 8 0 - 9 5 % ; whereas, 15(S)-methyl PGE I and ABPP each reduced the thymocytes 5 0 - 8 0 % . Although ABPP increased the spleen weight, the number of recoverable cells per spleen was no larger than that of the saline-treated controls. Cyclophosphamide, Ara-C, and 15(S)-methyl PGE I each reduced the number of recoverable cells/spleen and reduced spleen weights. Medroxyprogesterone acetate, a drug thought to have no immunomodulatory action, did not alter either the spleen weights or number of thymocytes. There was no difference between the fluorescence polarization P of DPH-labeled thymocytes from CBA mice treated with HCA and from saline-treated controls (Table I). The esr spectra of 5-NS-labeled thymocytes were typical of the fast anisotropic motion of the spin label generally observed in lipid bilyaers (Figure 2). The parameter 2Am~ is a simple measure of membrane fluidity: an increase in this parameter indicates a decrease in fluidity or. conversely, an increase in membrane rigidity. The values of 2Am~ for 5-NS-labeled thymocytes Table I

Fluorescence polarization at 3 7 ° C of DPH-labeled thymocytes from C B A mice treated with H C A or saline Polarization pa Experiment

I 2 3 X

H C A -Treated

0.233 0.217 0.227 0.226

-+ 0.016b +- 0.014b ~- 0.011b -+ 0.008

Saline

0.228 0.226 0.231 0.228

_+-0.008 ± 0.011 -'- 0.005 -* 0.003

aThe value of P reported is the mean of replicate measurements on a single labeled cell preparation; lhe reported precision is the standard deviation. bNot significantlydifferent from saline treated.

Membrane Fluidity of Murine Lymphocytes

55

PH

~IOG

t

2Amax Figure 2 Esr spectra of 5-NS labeled thymocytes from (A) HCA- and (B) saline-treated CBA mice. DPPH (2,2 diphenyl-1-picryl-hydrazyl free radical, Aldrich) was used as a reference. The scale in gauss (G) is indicated. derived from adult CBA mice treated with HCA, methylprednisolone or cyclophosphamide were larger than those for cells from saline-treated mice in nearly every experiment (experiments i - 9 , Table 2). This difference was noted in at least two of three experiments for all three drugs in the CBA mice and for HCA in AKR, C57BI/6, and B6CBF I mice (data not shown). The larger value for 2Amax of thymus cells from drug-treated animals indicates that these cells have a more rigid membrane than thymoeytes from saline-treated animals. This suggests that the immature thymocytes (> 90% of the cells from the saline-treated mice) have more fluid membranes than mature (CR) thymus cells. This hypothesis was supported by the smaller 2Amax of spin-labeled thymocytes from 6-day old animals relative to that of adult thymocytes in two of three experiments (experiments I 0 - 1 2 , Table 2). These young animals were assumed to have fewer mature CR thymocytes than the 6-to-9-week-old animals. Spin-labeled nucleated spleen cells from HCA-treated mice also had a larger 2Am~ than those from untreated mice (experiments 1 3 - 1 4 , Table 2). This indicates that. like CR thymocytes, CR splenocytes have more rigid membranes than CS cells. We found that thymus cells subjected to the ammonium chloride treatment used to lyse the erythrocytes in the spleen preparations had the same 2Am~ as the original suspension (data not shown); consequently, ammonium chloride lysis probably does not alter the splenocyte membrane. The cholesterol/phospholipid molar (C/PL) ratio was determined for each drug in both spleen and thymus cells. Treatment with any of the immunomodulatory drugs increased the C/PL ratio in the thymus cells as shown in experiments I, 3, and 4 in Table 3. Medroxyp-

Ceils

T

T

T

T

T

T

T

I

2

3

4

5

6

7

Adult

Adult

Adult

Adult

Adult

Adult

Adult

Mouse °

I0 94

15 134

9 98

10 IIi

8 125

10 125

13 120

Cells Recovered per Mouse X 100

methylprednisolone saline

cyclophospharnide saline

cyclophosphamide saline

saline

cyclophosphamide

HCA saline

HCA saline

HCA saline

Treatment

8 7

8 8

8 8

8 8

6 6

6 6

9 9

Number of Replicate esr Samples

52.2 ± 0 2 5 1 7 ÷ 0.3

52.0 + 0.3 51.4 ± 0.3

51.3 ± 0.2 51.1 ± 0.3

51.9 _+ 0.3 51.6 -+ 0.5

52.6 ± 0.5 51.9 ± 0.2

52.5 ± 0.4 52.1 + 0.4

54.6 ± 1.6 51.8 -+- 0.4

2Amaxb Gauss

yes

yes

no

yes

yes

no

yes

Different? c

2A .... values at 37°C for 5-NS-Labeled Thymus (T) and Spleen (S) cells from CBA Mice Treated with Immunosuppressive Drugs.

Experiment

Table 2

3.

T

T

T

T

S

S

9

I0

II

12

13

14

Adult

Adult

Adult 6-day old

Adult 6-day old

Adult 6-day old

Adult

Adult

40 90

101 128

40 22

133 15

72 9

12 105

9 142

HCA saline

HCA saline

none none

none none

none none

methylprednisolone saline

methylprednisolone saline

7 7

16 I0

I0 14

12 12

3 4

8 I0

8 8

51.3 _+ 0.4 51.0 +- 0.3

51.9 ± 0.3 51.7 ± 0.3

51.3 + 0.4 51.0 +- 0.4

51.7 ± 0.3 51.3 _+ 0.3

51.3 ± 0.2 51.0 ± 0.9

51.5 -* 0.1 51.0 ± 0.3

52.3 ÷ _ 0.2 51.9 ± 0.2

yes

yes

yes

yes

no

yes

yes

UCBA mice were 6-9 weeks old unless otherwise indicated. bThe value for 2Arnax is the mean obtained from the replicate .samples. The precision is reported as the standard deviation. CAre the two groups in the experiment significantly different: Yes means that the values of 2Amax are significantly different, p ~ 0.05; no. p > 0.05 using the Student t-test.

T

8

',,,.I

~c

R

= I~

o ~_

~. m,

"-n

~ E~ o"

58

M.V. Merritt et al.

Table 3

Molar Ratio of Cholesterol/phospholipid ( C/PL ) in Extracts of Thymus (T) or Spleen (S) Cells from CBA Mice Treated with Drugs or Saline

Cell Type

Drug

Cells recovered per mouse × 10 .6

I

T

saline HCA

140 8

0. 35 _+ 0.06 077 +_ 0.07

yes

2

T

130

0.45 _+ 0.I0

no

134

0.47 ± 0. I0

saline HCA cyclophosphamide

94 3 15

0.44 _+ 0.04 0.81 _+ 0 2 0 0.60 -~ 0.05

saline HCA cyclophosphamide

49 31 14

0.44 +_ 0.09 0.53 -+ 0.05 0.47 ± 0.09

yes no

saline HCA cyclophosphamide Ara-C 15(S)-methyl PGE I ABPP

57 5 3 9 16 I0

0.42 1.00 0.95 0.63 0.51 066

- 0.07 _- 0.02 + 0.13 -+ 0.06 -+ 0 0 6 + 0.08

yes yes yes yes yes

saline HCA cyclophosphamide Ara-C 15(S)-methyl PGE I ABPP

25 ii 3 12 13 25

0.37 0.43 0.46 0.39 0.27 029

± 0.04 _+ 0.03 + 0.03 -+ 0.04 _+ 0.06 ± 0.03

yes yes no yes yes

Experiment

3

T

S

4

T

S

saline medroxyprogesterone acetate

C/PL °

Is Drug b Different from Saline-Treated

yes yes

aThe value of C/PL reported is the mean of replicate determinations of lipid extracts of the pooled thymus or spleen cells from 7-13 animals per group, expressed as the molar ratio. The precision is reported as the standard deviation. bls the drug-treated group different from the saline-treated controls: Yes means that the values of C/PL are significantly different, p ~ 0.05: no, p > 0.05 using the Student t test.

rogesterone acetate, which does not alter immune function, had no effect on the C/PL ratio (experiment 2). HCA, cyclophosphamide, Am-C, and ABPP treatment increased the C / P L ratio of thymus cells in two additional experiments (not shown). Although 15(S)-methyl PGE I treatment increased the C / P L ratio slightly in experiment 4, in these additional experiments, the increase was not statistically significant. Drug treatment altered the C / P L ratio less in spleen cells than in thymocytes (Table 3). HCA treatment nearly d o u b l e d the C / P L ratio in recovered thymocytes, but increased this ratio in the spleen cells only slightly. C y c l o p h o s p h a m i d e also increased the C/PL ratio in spleen cells in o n e of two experiments s h o w n (experiments 3 a n d 4) and in o n e additional experiment (not shown).

Ara-C had no effect on C/PL of spleen cells in three separate experiments (one experiment shown). Both 15(S)-methyl PGE I a n d ABPP d e p r e s s e d the proportion of cholesterol in the spleen cells in experiment 4 (Table 3) but not in two additional experiments (not shown).

Membrane Fluidity of Murine Lymphocytes

59

DISCUSSION

This study compared the physical and chemical properties of lymphocyte membranes from mice treated with a variety of immunomodulatory drugs by three different methods. The effects of two immunosuppressive drugs, HCA and cyclophosphamide, have been studied previously by others; we have studied immunomodulatory drugs with different actions as well. In another paper, we will describe the effects of these drugs on thymus and spleen lymphocyte subpopulations defined by differentation antigens Thy-1, Lyt-1, Lyt-2 and surface IgG (submitted). Our final goal is to correlate the effects of the drugs on lymphocyte subpopulations with effects on the immune response. Glucocorticosteroids inhibit both humoral and cell-mediated immune responses when administered before antigenic stimulation, but are less effective when administered after antigen (Berenbaum, 1975). Their immunosuppressive activity is mild, however, considering the remarkable lymphoid depletion they cause (Blomgren and Anderson, 1971; Babu and Sabbadini, 1977; Frenkel and Havenhill, 1963), perhaps because mature thymocytes, immunocompetent peripheral T-cells, and B-cells stimulated by antigen (Blomgren and Anderson, 1971; Cohen et al., 1970; Babu and Sabbadini, 1977) resist destruction by steroids. Methylprednisolone is more potent than HCA, both as an immunosuppressive and as an antiinflammatory agent (Rosenberg and Lysz, 1980). Both cyclophosphamide, an alkylating agent, and Ara-C, whose triphosphate is an antimetabolite, inhibit cell proliferation (Berenbaum, 1975), but they clearly act very differently. Ara-C suppresses both antibody formation (Gray et al.. 1968a) and cell-mediated immunity (Gray et al., 1968b). Cyclophosphamide has a more complex constellation of activities: it suppresses antibody responses almost completely when given with antigen and induces tolerance (see Shand, 1979, for review), but under some circumstances can enhance delayed-type hypersensitivity (Askenase et al., 1975). Most cells which remain in the thymus after cyclophosphamide treatment are immunocompetent medullary thymocytes (Dumont, 1978; Turk et al., 1972; Dumont and Barrois, 1975) like the cortisone-resistant cells. Cyclophosphamide and HCA, however, do not affect thymocytes identically. Using size and electrophoretic mobility, Droege et al. (1974) showed that one subpopulation of thymus cells sensitive to cortisone was resistant to cyclophosphamide treatment. Cyclophosphamideresistant murine spleen cells, like CR spleen cells, appear to suppress the generation of antibodies to a thymus-dependent antigen (Mclntosh et al.. 1979). Prostaglandins of the E type have a variety of immunomodulatory activities (Goodwin and Webb, 1980). 15(S)-methyl PGE I modulates both immunological and inflammatory reactions. It affects antibody formation to sheep erythrocytes in mice very little, but it alters the GvH reaction; given on days 1 - 3 of a 10-day reaction, it enhances splenomegaly, but given on days 7 - 9 , it reduces splenomegaly significantly (P. E. Fast, unpublished data). This compound inhibits the reversed passive cutaneous Arthus reaction in the rat, an inflammatory reaction initiated by immune complexes and complement, as well as the release of lysosomal enzymes and oxygen-derived free radical generation by polymorphonuclear leukocytes (Kunkel et al., 1979; R. J. Smith and S. S. Iden, personal communication). It also delays the death of NZI3/W (Zurier et al., 1980) and MRL-Ipr/IprI mice from immune complex glomerulonephritis. The compound ABPP is an isocytosine analogue which induces interferon and also seems to protect against viral infections by a stimulation of host defenses which may be independent of interferon (Stringfellow and Weed. 1980: Renis and Eidson, 1980). At the dose used in the experiments described here. ABPP increases antibody-forming cells (against sheep red blood IMcllroy, W. and P.E. Fast. Presented at the First Internal~onal Conference on Immunopharmacology, Brighton, England. August, 1980.

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M.V. Merritt et al.

cells) in both immunized and unimmunized mice (Fast and Stringfellow, 1980; Fast et al., 1979a). It also increases the natural killer activity (Loughman et al., 1980). On the other hand, this compound inhibits the response to T-cell mitogens (Taggart et at., 1980) and the GvH reaction (Fast et al., 1979b). We found that treatment with any of the drugs of this study (except medroxyprogesterone acetate), despite the diversity of their in vivo immunological effects and proposed mechanisms of action, altered the C/PL molar ratio in thymocytes. The lipid composition data following HCA treatment reported here agree with the previous work of Kigoshi (1978). These data indicate a relationship between thymocyte membrane cholesterol content and immunological competence. We found that other immunologically active drugs also increase the C/PL ratio in thymocytes; whereas, only HCA and cyclophosphamide significantly alter this ratio in the cells of the spleen. The rather marked alterations in the thymic C/PL ratio caused by HCA and cyclophosphamide are probably due to the loss of many cells from the thymus. We could recover only about 5% as many thymocytes from mice treated with these drugs as from saline-treated controls. The C/PL ratios of the thymocytes from animals treated with Ara-C, 15(S)-methyl PGE], or ABPP are intermediate between those of the cells from saline and from HCA- or cyclophosphamide-treated animals, as are the numbers of recoverable thymocytes. It is likely that we are measuring pre-existing properties of the drug-resistant cells, but we cannot rule out drugqnduced alterations in membranes. Most of the cells were probably killed by the drug treatment, and some may have migrated to other lymphoid tissue (Claman, 1972; Cohen et at.. 1970). Studies of cell size, density and electrophoretic mobility have shown that small dense thymocytes with slow electrophoretic mobility disappear from the thymus after corticosteroid treatment (Droege et al., 1974). These cells are thought to be immature or immunoincompetent, although they are not present at birth. Corticosteroids eliminate, from the thymus. cells of medium to large volume with intermediate electrophoretic mobility, but not large medullary thymocytes, which are thought to be immunocompetent cells ready to leave for the peripheral organs. Our flow cytometry data (submitted) indicate that treatment of mice with Ara-C, cyclophosphamide or ABPP also reduces the number of small cells in the thymus as has been previously shown for HCA (Mathieson et al., 1979; Ledbetter et al., 1980). Spin labeling data indicate that the thymocytes isolated from mice treated with HCA, cyclophosphamide, and methylprednisolone have more rigid membranes than control thymocytes, as determined by the larger value of 2Areax, and that membranes of spleen cells remaining after HCA treatment are more rigid than controls. A principal feature of the fluid-mosaic model of biological membranes (Singer and Nicolson. 1972) is the mobility of lipid and protein. The ease of motion in the membrane bilayer is often expressed as the degree of fluidity, a property that is the reciprocal of viscosity (Hildebrand and Lamoreau, 1972). A certain degree of membrane fluidity appears to be crucial for many membrane functions; furthermore, cholesterol modulates membrane fluidity (Chapman, 1975). Spin labeling and fluorescence polarization studies have shown that mitogenic lectins temporarily increase lymphocyte membrane fluidity (Toyoshima and Osawa, 1976; Barnett et al., 1974). Increasing the cholesterol content of the lymphocyte membrane blocks both the change in fluidity and activation by lectins (Toyoshima and Osawa, 1976; Alderson and Green, 1975). On the other hand, Heiniger et al. (1978) have shown that cholesterol synthesis and, by implication, increased cellular cholesterol content, is necessary for T-lymphocyte cytotoxicity. Yashuda and coworkers (1977) have demonstrated that the phospholipid composition markedly influences the immunogenicity of model membranes. An examination of the spin labeling literature indicates that the small difference between 2AreaX ( 1 - 2 % ) of thymocytes of HCA treated mice and saline controls is less than might be expected from the two-fold difference in the C/PL molar ratio. Boggs and Hsia (1973) examined the effect of cholesterol content on the structural organization of a variety of lipid

Membrane Fluidity of Murine Lymphocytes

61

classes by esr spectroscopy of planar films labeled with 5-nitroxide palmitate. Increasing the mole percent of cholesterol from 0 to 33% in phosphatidylcholine films, they found a 4.8% increase in an esr parameter proportional to 2Am~; they observed, under identical conditions, a slightly smaller increase in this parameter in phosphatidylethanolamine films. Using a spin labeled steroid, Lapper et al. (1972) examined the esr spectra of hydrated egg lecithin mullilayers as a function of cholesterol content. They found that 2AreaXincreased approximately 4% for an increase from 30 to 45 mole percent cholesterol. Their study mimics the differences between the cholesterol content of the thymocytes from controls and that of the HCA-treated mice in our experiments: the thymocyte lipids from the saline-treated controls contained 30 mole percent cholesterol; whereas, those from CR-thymocytes contained 45 mole percent cholesterol (calculated from the sum of the cholesterol and phospholipid content). The smaller difference in 2Am~ we have observed using 5-NS labeled cells may be attributable solely to the different spin labels used. Our inability to detect any differences in membrane fluidity between thymocytes from HCAand saline-treated animals by fluorescence polarization measurements was at first somewhat surprising since the differences in C/PL ratios were so large. Comparison of our data with other DPH fluorescence polarization studies suggests that we should have observed a difference in P. Kalra (1977) has compared the value of P with the C/PL ratio of lymphocytes from pigeons susceptible and resistant to atherosclerosis. He found that the value of P of DPH-labeled cells and the relalive proportion of cholesterol were both 2 0 - 2 5 % lower in the lymphocytes from susceptible birds. Shinitzky and Inbar (1976) have examined various fluorescence parameters of DPH as a function of cholesterol content in model membrane systems. They found that P increased from 0.20 to 0.26 in phosphalidylcholine liposomes as the molar C/PL ratio was increased from 0.4 to 1.0 at 25°C. We are unaware of a previous report in which both the DPH fluorescence probe and the 5-NS fatty acid spin label were used to evaluate the effect of a pharmacological agent on the membrane fluidity of whole cells. Consequently it is difficult to make a direct comparison of our contradictory results with those of another study. We can, however, examine separate studies in which only one of these probes were used to examine the same system. Harris and Schroeder (1981) recently reported that 20mM ethanol produced a 5% decrease in the polarization of DPH fluorescence in mouse brain synaptosome membranes. In contrast, Chin and Goldstein (1977) found this same ethanol concentration yielded a decrease of only I - 2 % in the order parameter S, a parameter related to 2AreaX, of synaptosomal membranes labeled with 5-NS Harris and Schroeder concluded that these differences indicated that low, physiologically relevant concentrations of ethanol selectively fluidize the hydrophobic core of synaptic membranes. This conclusion was based upon the knowledge that DPH selectively partitions into the hydrophobic lipid core of the membrane bilayer; whereas 5-NS is a probe of more superficial membrane regions. By analogy our results would suggest that the drug-resistant lymphocytes may be less fluid near the membrane surface than the controls. There are, however, alternative explanations for the contradictory fluorescence and spin labeling results. The cholesterol distribution may be nonuniform in the plasma membrane (Demel et al., 1977) or the membrane fluidity may be modulated by components other than cholesterol. It has been shown, for example, that the decrease in fluidity of rat adipocyte membranes with maturation can be attributed to an alteration in the degree of saturation of the phospholipid fatty acids (Hubbard and Garratt, 1980). We are currently examining the phospholipid composition of the drug-resistant lymphocytes and the plasma membrane of these cells to evaluate its relationship both to fluidity and to immunocompetence. Our data indicate that, of the physical/chemical techniques examined, direct chemical analysis provides the greatest information about the effects of immunomodulatory drugs on lymphoid cell populations. In another report, we will show that the cell size and expression of the Lyt and Thy-1 surface antigens can be correlated with the relative cholesterol content of murine thymocytes.

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We thank E.L. Sun and K.A Annis for help in preparing cell suspensions, and C.L. Franz and SK. Moyer for editorial assistance. REFERENCES

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