Activated macrophages kill tumor cells independent of membrane fluidity

Vol.

89,

No.

July

27, 1979

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

ACTIVATED MACROPHAGES KILL INDEPENDENT OF MEMBRANE

Stephen

W.

Russell?

and

520-525

TUMOR CELLS FLUIDITY

Alfred

F.

Esser*

t

Department of Pathology University of North Carolina-School of Medicine shape1 Hill, North Carolina 27514 and Department of Molecular Immunology Research Institute of Scripps Clinic La Jolla, California 92037 Received

June

4,

1979

SUMMARY: By using electron spin resonance techniques and 5-doxyl stearate spin labels, we have measured the apparent membrane fluidities of several kinds of macrophages. The tested cells included non-cytolytic thioglycollate-elicited peritoneal macrophages and either cytolytic or non-cytolytic macrophoges isolated from regressing Moloney sarcomas. Our results indicated no characteristic plasma membrane fluidity that distinguishes killer macrophages from their non-killing counterparts. Furthermore, no relationship existed between the generalized plasma membrane fluidities of activated macrophoges and the neoplastic target cells that they killed.

Macrophoges con

kill

tumor

however,

(1,2).

The

it is non-phagocytic effector

somehow

distinguishes

Hibbs

(5,6)

plasma

and

cytolytic

(regressor

target

activity

macrophages However,

kill

such

can

neoplastic minute

amounts

and neoplastic “recognition”

hypothesis

be

modulated

cells

but

after

(pg-to-rig/ml)

0006-291X/79/140520-06$01.00/0 Copyright @ I979 by Academic Press, Inc. All rights of reproducrion in any form reserved.

killing

(or

extremely

from

cells

our

close the

and result

kills

24 h --in vitro of bacterial

“activated” is not

only

known;

juxtaposition)

activated

of similar

the

mocrophage latter

fluidities

(3-5). in the

ccl Is. recently

spontaneously

predictably

520

process

Apparently,

is the

with

is colled

in the

and transformed

directly isolated

definition,

contact (3,4).

macrophages

of mocrophages

macrophages)

involved and

normal

of activated this

of a clearer

is required

that

to test

lock

mechanism

cells

between

membranes

for

in nature,

has postulated

We decided

(7).

that,

celis

between

the

in o state

developed

system

regressing

Moloney

in

which

sarcomas

and

consistently.

Freshly

explanted

lose

their

activity

entirely

(LPS,

endotoxin)

cytolyfic

lipopolysaccharide

Vol.

will

89,

trigger

elicited LPS

No.

the

BIOCHEMICAL

sudden

peritoneal

resurgence

macrophoges

AND

of high cannot

BIOPHYSICAL

levels

of this

be stimulated

RESEARCH

killing

activity

to kill

COMMUNICATIONS

(8).

by similar

Thioglycollate-

concentrations

of

(8). Electron

spin

search

for

This

technique

neoplastic

vated activated

isolated

(i)

(ESR)

relationships

has been

target

that:

resonance

possible

macrophages

show

2, 1979

cells There

macrophages, macrophages

spectroscopy,

between

employed

from

tumors

by

exposure

cytolytic

recently

No relationship

and the

neoplastic MATERIALS

acid

that

amounts

plasma

the

plasma

when

fluidity

between

cells

that

they

AND

METHODS

labels,

the

cells The which

plasma

was

membrane

membrane

these

of LPS (9).

membrane

exists

spin

and plasma

perturbed

to minute

(ii)

fatty

activity

to show

is transiently

is no characteristic and

using

used

to

fluidity.

of non-cytotoxic are triggered

results

we report

distinguishes membrane

to kill here acti-

fluidities

of

kill.

Mice and Tumor Induction: BALB/c AnCr male mice aged 6-10 weeks were sarcomas, which previously have been shown to r;gress spontaneously (lo), were intramuscular (gastrocnemius) injection of 5 x 10 cultured MSC rhabdomyosarcoma days earlier (11).

used. Moloney induced by the cells eleven

Tumor Disaggregation and Isolation of Macrophages: Tumors were minced and disaggregated enzymatical ly using a mixture of trypsin, collagenase, and deoxyribonuclease (12). Macrophages welre isolated in monolayers on the bottoms of 16 mm-diameter, flat bottomed, plastic wells, as described (7,8). The plating procedure yielded on adherent population of cells that numbered 1.5 x 106/weII, as determined by direct counting of cells washed from wells. Their appearance and ability to phagocytose zymosan particles established that 90 to 95% were macrophages. The other cell type consistently found in these monolayers -- usually ~5% but sometimes up to ‘IO% of the total -- was the MSC cell used to initiate the sarcomas. Monolayers held in culture for 24 h were incubated in HEPES-buffered (15 mM) Eagle’s minimum essential tissue culture medium (H-MEM) containing 30% fetal bovine serum (FBS). Thioglycollate was used to elicit peritoneal macrophages ond they were harvested os previously described (8). Monolayers (containing >95% macrophages were established by allowing peritoneal exudate cells to settle either on the bottoms of plastic wells or on the inner surfaces (front and back) of glass “microslides” (Vitro Dynamics, Rockaway, N J). Non-adherent ccl Is were removed from these monolayers by repeated, vigorous washings. LPS and Assay of Macrophage-Mediated Cytolysis: Purified LPS isolated from Escherichia col i -4) was obtoined from Dr. David C. Morrison of the Research Institute of Scripps Clinic. The preparation and quantification of this reagent have been described (8). As before (7,8), the amount of chromium-51 (5TCr) released from prelabeled P815 mastocytoma cells in 16 h was used to indicate macrophage-mediated cytolysis. Spin Labeling of Plasma or jetted free (Pasteur pipette)

Membranes: from their

Adherent substratum,

521

cell

types washed

were scraped (rubber policeman) by centrifugcfion (5 min, 4O,

Vol.

89,

No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNKATIONS

Figure 1. ESR spectra for the two different spin labels in the membranes of macrophages The outer peaks are retraced at a higher sen-from regressing Moloney sarcomas. Rate of decay of the sitivity setting to facilitate accurate measurements of 2Tll values. Instrusignal at this temperature is indicated by the sloping trace at the top of the figure. ment settings: Modulation amplitude, 2.0 G; microwave power, 10 mW; temperature, 21°C.

600 gmax) and resuspended in 100 pl of ice cold PBS. The final cell concentration was Spin labels (either 5- or 12-doxyl stearate from SWA, Palo Alto, CA) usually 2 x 107/mi. were dissolved in ethanol (125 pg/ml). S even microliters of this solution were added to the chilled cell suspension. After 10 min the cells were diluted to 2 ml with ice cold PBS and pelleted as before. After two additional washes at 4” the cells were resuspended in 70 pl of PBS and sealed into glass capillary tubes. The viability (trypan blue dye exclusion) of culTumor macrotured cells or macrophages jetted free from their substratum exceeded 95%. phages scraped from plastic wells were 70 to 85% viable, but tightly adherent, thioglycollateelicited peritoneal macrophages treated similarly were rarely more than 50% viable. Because of the latter’s relatively low viability, in some experiments thioglycollate-induced macrophages were cultured and analyzed Under these conditions >95% of --in situ on microslides. the thioglycollate-elicited, adherent macrophages remained viable throughout the Idoeling and examination processes. ESR equipped 535 signal splitting or by the ence (9).

spectra were recorded with a Varian (Palo Alto, CA) model E-104 spectrometer with a temperature controller and interfaced with a Nicolet (Madison, WI) model averager. Sample temperatures were measured as described earlier (13). Hyperfine parameters were determined either from the computer averaged and expanded spectra method of Gordon and Sauerheber (14). For further details see Figure 2 of refer-

RESULTS Figure difference

1 shows

that

the

spectra

denotes

that

the

labels

AND for

were

the

DISCUSSION 5-doxyl

located

and within

12-doxyl a flexibility

stearate

were

gradient

different. in a membrane

This

Vol.

89,

No.

2, 1979

HYPERFINE

BIOCHEMICAL

SPLITTING MOUSE

(2T,,)

AND

TABLE

I

OF

5-DOXYL

VALUES

MACROPHAGES

BIOPHYSICAL

(Ma)

AND

RESEARCH

STEARATE

NEOPLASTIC

IN

CELL

COMMUNICATIONS

MEMBRANES

OF

LINES

CELLS

2TI,

(GAUSS)* 37oc

21°C NONCYTOLYTlCt

MACROPHAGE:

1. 2.

(isolated

Thioglycollate-elicited Cultured Ma Maloney

sarcoma

CYTOLMICt

peritoneal M@ from regressing held 24 h in culture)

and

55.2

* 0.3

51.9

* 0.5

54.8

f 0.4

51 .o f 0.5

MACROPHAGE:

1.

Freshly

explanted

2.

sarcoma Cultured

M@

(100

stimulated

rig/ml,

Maloney

with

CELL

51.3*<0.1

LPS

16 h)

TARGET P815 MSC

regressing

55.2rtO.3

NEOPLASTIC 1. 2.

from

55.0

+ 0.3

51.5

+ 0.5

56.0 53.8

i 0.2

51.8 50.8

i 0.2

LINES:

mastocytoma rhabdomyosarcoma

* ,Mean i standard deviation of four or more separate determinations. ‘Values without standard deviations are of single determinations. t ‘Cytolytic capacity was determined by 5’ Cr release from target cells as described under Methods. Release of 51 Cr, using P815 mastocytoma and MSC rhabdomyosarcoma ‘cells as targets in a 16 h assay wa5 in the range of 32 to 50%.

rather the

than spin

merely

labels

adsorbed

were

ferricyanide

ions

membrane

perturbation

likely

that

the

Chonges in the can

be used

reduced

can

that

fatty

acid

rotation

to assess this

of the

lipid

environment

branes

of macrophages,

surfaces

(15).

Also

shown

cells.

As we

have

described

by the

reoxidize

in distance

anisotropic

to cell

almost can

spin

labels.

be recorded labels

between of the group’s in which 5-doxyl

all

ore the

by such confined

outer

of motion

showed

223

2Tl,

(9),

membrane

the These plasma

maxima

(2T,,

group. and,

(15).

1 is the

the

nitroxyl

it is located stearate

labels.

within

hyperfine

spin-carrying freedom

In addition,

in Figure

thus,

two

facts

impermeant LPS produces

make

value)

it very

reflect the

to characterize

indicative

at which

membrane.

Therefore,

Wh en incorporated values

impermeont

rate

into

alterations 2T the

the

II

rigidity

plasma

of a moderate

value

mem-

viscosity.

a

Vol.

89,

No.

2, 1979

Populations measured found fact

at 21 o and

that

obtained

sured

the differed

from

the

were I,

Thus, on the

basis

target

cells.

selectivity

that

in terms

of

no clear

by

and

neither

(Table

to kill

I).

No

macrophage shown

in

neoplastic

target

cell

lines.

of these

macrophages fluidities

cell

types

were

differences

were

in spite

I).

values (7),

However,

membranes

values

of the

2T,,

As expected

plasma

2Tll

cells

Table I are

(Table

of their had

neoplastic

populations,

Also

in the

COMMUNICATIONS

important

another.

activated

was found

RESEARCH

capacity

various

one

different

to killing

similarity

stearate

2Tl I values

from

of two

BIOPHYSICAL

in their

of these

functionally

analyses

AND

differed

membranes

susceptible

by 5-doxyl

of cytolytic

37’

plasma

they

cells

in Table

may

of macrophages

between

these

BIOCHEMICAL

that

asshown as mea-

resembled

those

macrophages. the

selective

killing

of generalized These of the

be responsible

similarities

results killing for

of tumor

argue process

macrophage-mediated

by

between

against and

cells

the

should

activated the

theory serve killing

macrophages

cannot

fluidities

of the

membrane that to shift

Hibbs

(6)

attention

of tumor

has put forth to other

be explained effector

and

to explain

mechanisms

the that

cells.

ACKNOWLEDGEMENTS Thanks are extended to Ms. Priscilla Munkenbeck for her technical was supported in part by USPHS research grants CA 23686 and AI 14099. of Research Career Development Award CA 00497 and AFE of Established 76-225 from the American Heart Association. This is publication number Research Institute of Scripps Clinic.

assistance. The work SWR is the recipient Investigatorship 1713 from the

REFERENCES 1. 2. 3. 4. 5.

Hibbs, J.B., Jr., Lambert, L.H., Jr. and Remington, J.S. (1972) Nature New Biol. 235, 48-50. Keller, R. (1976) in: Immunobiology of the Mocrophage (Nelson, D.S., ed) pp. 487-508, Academic Press, New York. Meltzer, M. S., Tucker, R.W. and Breuer, A.C. (1975) Cellular Immunol. 17, 30-42. Bucana, C., Hoyer, L.C., Hobbs, B., Breesman S., McDaniel, M. and Hanna, M. G., Jr. (1976) Cancer Res. 36, 4444-4458. Hibbs, J.B., Jr. (1974) Science 184, 468-471.

524

Vol.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

89,

No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Hibbs, J.B., Jr. (1976) in: The Macrophage in Neoplasia (Fink, M.A., ed) pp. 83-111, Academic Press, New York. Russell, S.W., Gillespie, G.Y. and McIntosh, A.T. (1977) J. Immunol. 118, 1574-1579. Russell, S.W., Doe, W.F. andMcIntosh, A.T. (1977) J. Exp. Med. 146, 15111520. Esser, A.F. and Russell, S.W. (1979) Biochem. Biophys. Res. Commun. 87, 532540. Russell, S.W., Gillespie, G.Y., H ansen, C. B. and Cochrane, C. G. (1976) Int . J. Cancer 18, 331-338. Massicot, J.G., Woods, W.A., Chirigos, M.A. (1971) Appl. Microbial. 22, 1119-1122. Russell, S.W., Doe, W.F., Hoskins, R.G. andcochrane, C.G. (1976) Int. J. Cancer 18, 322-330. Esser, A.F. and Lany, J.K. (1973) Biochemistry 12, 1933-1939. Gordon, L.M. and Sauerheber, R.D. (1977) Biochim. Biophys. Acta 446,34-43. Hubbell, W.L. and McConnell, H.M. (1971) J. Amer. Chem. Sot. 93, 314-326

525