CELLULAR

IMMUNOLOGY

20, 12-24 (1975)

Studies on the Effect (Lymphokines) I. Early Changes

L. W.

of Soluble

Lymphocyte

on Macrophage in Enzyme Activity POULTER

AND

J. L.

Physiology and Permeability TURK

Royal College of Surgeons of England, 35-43 Lincoln’s London WCZA 3PN, England Received

Products

Inn Fields,

February 24,1975

Various cytochemical techniques have been used to quantitate the rapid effect of a partially purified, soluble product from lymphocytes (lymphokine) on normal guinea pig macrophages in vitro. Early changes in the utilisation of hydrogen liberated from the hexose monophosphate shunt and on cellular permeability were observed. The ability of the lymphokine to alter hydrogen utilisation was also seen in experiments on cryostat sections of guinea pig liver, suggesting that the cytochemical effects were not predetermined by changes at the membrane level. It is suggested that lymphokine-induced changes within the cell may reduce some biosynthetic activity affecting the cell membrane and this may in part reflect the decreased migrating ability of the cells. Increases in NADPH oxidation after lymphokine contact are discussed in relation to the bactericidal capacity of the cells.

INTRODUCTION It has been established that soluble products from sensitized lymphocyte-antigen interaction are capable of affecting macrophage function in vitro (l-4). These factors have been collectively termed “lymphokines” (5) and among their effects are the inhibition of macrophage migration (for reviews see (Ref. 1, 2) ) and the biochemical activation of these cells (3, 4). Although these changes occur after different times of contact, it has been suggested that they are produced by the same factor derived from sensitized lymphocytes and that the early changes in macrophages, which result in migration inhibition, render the cells receptive to activation at a later state (4). It was previously reported (6) that the cytochemical and morphological effect of mediator-rich fractions on macrophages was biphasic. The initial phase (up to 5 hr) resulted in an apparent decrease of hexose monophosphate shunt (HMPS) activity and a rounding up of the cells. This was followed by a second phase that ultimately resulted in the biochemical activation of the cells. Inhibition of spreading (7), decreased macrophage volume (8)) and increased adhesiveness (3, 4) have also been reported as effects of lymphokines. Whether there is a relationship between these physical changes and the effect of mediators on macrophage cytochemistry, which eventually results in activation, has yet to be determined. 12 Copyright 1975 by AcademicPress,Inc. All rights o9 reproduction in any form reserved.

RAPID

EFFECTS

OF LYMPHOKINE

ON MACROPHAGES

13

This paper examines a variety of cytochemical parameters to see whether such a relationship does exist. In doing so, early effects of lymphokine on cultured macrophages are observed that suggest changes, in particular of HMPS activity, as earl\ as 1 hr after contact. MATERIALS Prodllcfion

AND METHODS

of Lymphokine

Lymphokine-rich fractions were prepared by the method of 1Volstencroft and Dumonde (9). Lymph node lymphocytes from guinea pigs immunized 2 weeks earlier with 100 PLgof bovine y-globulin (BGG, Armour Pharamaceutical Co. Ltd., England) in Freund’s complete adjuvant (FCA, Difco Labs., Detroit, Mich.) were cultured for 24 hr in Eagle’s medium supplemented with 1 ml of glutamine, 200 pM; 1 ml of sodium pyruvate, 100 PM, 5 ml of 4.47 o sodium bicarbonate, lo4 units of penicillin, and 10 mg of streptomycin, all in 100 ml. The cell concentration was 5 X lo6 cells/ml and BGG was added at a concentration of 1 mg/ml. The supernatant fluids were lyophilised, dialysed, and purified on Sephadex G-100 to a molecular weight range of 25-75 x 103. Lymph node cells from the same animals were also cultured without the addition of BGG, the supernatant fluids being reconstituted with an equivalent amount of antigen after the incubation. These control supernatants were treated in an identical manner to the mediator-rich supernatant fluids. After purification the lymphokine-rich and control fractions were concentrated to a level where a 1 : 10 dilution of the lymphokine would inhibit normal macrophage migration by 50% compared with an equivalent concentration of the control fraction. Jlacrophagr

Cultawe

Peritoneal macrophages obtained from guinea pigs stimulated with sterile liquid paraffin 4 days previously were cultured in Leighton tubes as previously described (6). Peritoneal exudate cells, 2-3 x 106, were plated onto coverslips in Leighton tubes in 1 ml of Eagle’s medium containing 15% inactivated guinea pig serum. After l-2 hr of culture at 37°C nonadherent cells were washed off and the medium was changed for one containing 50% inactivated guinea pig serum. The gas phase was 10% CO2 in air. With the exception of one set of experiments (see Results) the cells were left in normal culture for 6 days. L,ymphokine or control fractions were added under sterile conditions after the 6 days normal culture so that the final concentration of the lymphokine was that which had been shown to cause 50% inhibition of migration. Experiwaentation

In initial experiments the coverslips were removed from the Leighton tubes after 2 hr or 6 days. In subsequent experiments lymphokine or control fractions were added to the cultures after 6 days and the coverslips were then removed after they had been in contact with the test fractions for 1 hr. At whichever time the coverslips were removed they were washed in Eagle’s medium and allowed to dry at room temperature. The coverslips were then cut into small pieces and the cytochemical reaction medium was immediately placed onto the cells while thev

14

POULTER

AND

TURK

were still on the coverslips, as previously described (6). The conditions for each cytochemical reaction are described below. Quadruplicate cultures were examined for each #test performed in each experiment and the mean and standard error recorded. As there was some variation in activity from one set of cultures to another, each comparative experiment was performed within one set of cultures on the same day. Throughowt the results no comparisons were made of absolute activity from one set of cultures to another set originating from a separate animal. All experiments were performed at least twice and the results given are of a typical experiment. In some experiments blocks jj cm3 were cut from normal guinea pig liver. These were frozen in hexane at -70°C (10) and cut into sections 10 pm thick on a cryostat. Cytochemical tests were performed on quadruplicate sections. Cytochemical Techniques Tetrazolium reduction has been shown to give comparable results to oxygen uptake in the biochemical estimation of dehydrogenase activity (11). Altman (12) has demonstrated that the cytochemical estimation of pentose shunt dehydrogenation yields comparable results to the biochemical assay of Gloch and McLean (13). In a later paper (14) a table of comparisons was published showing that similar activities to ‘those detected biochemically can be demonstrated for succinate dehydrogenase and glutamate dehydrogenase using these cytochemical techniques. 1. Dehydrogenases. The reaction medium to demonstrate dehydrogenase activity cytochemically was composed of 0.05 M glycylglycine buffer, pH 8.0, containing 20% polyvinyl alcohol (PVA grade B05/140, Cytochemical Co., Edgware, Middx.) to which was added a tetrazolium salt as the hydrogen acceptor, at a concentration of 3 mg/ml. The tetrazolium salts used in this study were MTT and NBT.l To demonstrate glucose-6-phosphate dehydrogenase (G-6-P.D.) , glucose-6phosphate disodium salt (Boehringer Corp., London) was added as the substrate at a concentration of 3 mg/ml and NADP (Boehringer Corp., London), also at a concentration of 3 mg/ml, was added as the coenzyme. In addition phenazine methosulphate (PMS, Sigma Chemical Co., London) was added to some of the reaction mixtures at a concentration of 40 rg/ml. Unlike the tetrazolium salts which can only be reduced by the liberated hydrogen when this has dropped to an appropriate electropotential PMS can trap the hydrogen directly from the coenzyme and then reduce the tetrazole nonenzymatically. One can therefore quantitate the total amount of hydrogen liberated by the G-6-P.D. (if PMS is used) or the amount of hydrogen passed along the endogenous NADPH oxidation pathway (no PMS used) (6, 14, 15). The difference between these two levels reflects the hydrogen liberated that is available for biosynthesis and, in particular, fatty acid biosynthesis (14). To demonstrate NADPH oxidation, NADPH disodium salt (Boehringer Corp., London) was used as the substrate at a concentration of 3 mg/ml. Succinate dehydrogenase activity (Succ. D) was demonstrated by using sodium succinate (BDH, Poole, Dorset) as the substrate at a concentration of 12.5 mg/ml. MTT was used as the hydrogen acceptor and PMS was always added to this reaction 1 MTT, 3- (4,Sdimethyl thiazoyl-2) -2,4-diphenyl bromide ; NBT, (3,3’-dimethoxy-4,4’ diphenylene) ditetrazolium chloride.

2,2-di-p-nitrophenyl-3,3-

KAPII) EFFECTS OF LYMI’IlOKINE TABLE

1;

OK ~I.\(‘KOI’1I~\(;l;: millilitw C-OIresponclin~

Dimethyl formamidc

550

.Mkaliw dimethyl formamideCz

6 75

_‘I

‘).A

ClAlkaline dimethylformamide was prepared by the addition of loci; (v/v) of a buffer, ptl 12, consisting of 0.1 Msodium hydroxide, SVC (0.1 M glycine $ 0.1 M sodium chloride). It is add4 10 the cells after the dimethylformamide.

at a concentration of 40 pg/ml. All reactions were performed at 37°C. Controls were performed omitting the substrate, except in the case of Succ. 11 activity where the specific inhibitor sodium malonate was used (10) and these levels of “activity” were subtracted from the results. 2. L~SOSON~~ actWit?. ,&Glucuronidase activity was used as a lysoso1n;1l enzyme marker. The activity was demonstrated using a postcoupling techniclue as tlescrib~tl elsewhere (lo), the only difference being the ommission of PVA. Naphthol AS-lZT,&glucuronide (Calbiochem T,td., I,ondon) was used as the substrate at a concelltration of 23 pg/ml and fast blue l3 salt (Serva. Heidelberg, Germanyj ivas 11~1 as the coupling salt at a concentration of 0.5 mg/ml. 3. Szclphydryl protein. To demonstrate concentrations of sulphydryl SH ;ul(l disulphide SS protein, an adaptation of the ferro-ferricyanide reaction (16) ~1s ~a5 used. A 3 : 1 mixture of 1% ferric chloride and 0.1% potassium ferricyanide added to the coverslips for three periods of 10 min each at room temperature. ‘I’I) determine the proportion of SS protein the macrophages were incubated in 20?$ sodium dithionite for 30 min at 37°C and washed in running tap water for 2 niin before staining with the ferricyanide mixture. This treatment reduces the SS groups to SH. The difference between the total staining ( SH + SS) and the Sf l alone was taken to represent the proportion of SS protein. Controls \vere performed by preincubating the macrophages for 4 hr in 0.1 .%I iv-ethyl 111ale:1111itle (17) which was dissolved in a 20% solution of PVX in distillecl xvxter and results \vere subtracted from the test readings.

In the dehydrogenase reactions the liberated hydrogen reduces the tetr;~zoliun1 salt to form an insoluble, coloured formazan, which was eluted from the macrophages on each piece of coverslip into 1 ml of solvent and its optical density (OI>j was measured on a spectrophotometer. The solvent used, the absorption maximum of the extracted formazan, its extinction coefficient (k’j , the molecular wright of

16

POULTER

NBT

NET .PMS

MTT

AND

MTT * PMS

TURK

SuccD

Succ 0.

l O.OSm Sodturn malonato

FIG. 1. Nonspecific absorption and reduction of NBT and MTT by cultured macrophages. The effect of sodium malonate (0.05 M) on Succ. D activity is also shown. Mean of quadruplicate cultures 2 SE.

the tetrazole (MW), and the moles of hydrogen required to produce 1 mole of formazan (N) are given for both the tetrazolium salts used (Table 1). After extracting the formazan the coverslips were washed in ether and the nucleic acid was extracted by the method of Butcher (18). The cells on the coverslips were incubated in 0.7 ml of 1 N HCl at 60°C for 30 min and the extracted nucleic acid was measured on a spectrophotometer at 265 and 310 nm. The 310-nm reading was subtracted from the 265-nm reading to give the optical density-nucleic acid (ODNA). The nucleic acid from lo6 cells extracted in this way into 0.7 ml was found to have an optical density of 0.92. The results were therefore expressed by the formula below: OD (formazan) MW

X K

X N X

0.92 =X

1000 = nmoles of hydrogen/lo6

cells.

The ferricyanide reaction and the /3-glucuronidase activity were quantitated on an The use of this machine has been previously integrating microdensitometer. described in full (19, 20). At least 20 areas of l-3 cells were scanned from each specimen and the relative absorption for each area was recorded. The units of relative absorption recorded by this machine are solely comparative. They allow the quantitation of changes in amoum of reaction product from one enzyme to another and from one time to another as this is reflected in the absorption of light over a given area but should not be regarded as absolute units of reaction product. All quantitative data were subjected to statistical analysis using Student’s t test for nonpaired data. RESULTS Cell Yield and Viability Of the 2-3 x lo6 cells plated onto the coverslips originally, many are lost during the culture period. After 6 days between lo5 and 4 X lo5 cells remain on the coverslips, however, as cell death results in the cells becoming detached from the glass the viability of the remaining cells was found to be 8084% as detected by trypan

KAPII)

EFFECTS

OF LYhf PHOKINE

OK

MrZ(‘ROPII.4(;ES

ti

200-

160. I‘Oy

T.

P=-o-1

120-

P>0.1 f-h

NADPH.0.

G-6-P.D

GMD.+PMS

succ.0.

FIG. 2. A comparison of the respiratory activity in normal guinea pig macrophages after 2hr culture (shaded) and 6-day culture. Mean and standard error are given. Results are from lo-min incubations except Succ. D which was incubated for 20 min. Values are mean * SE.

blue exclusion. Although only S-2070 of the original cells remain on the coverslips this number was found to be sufficient for quantitating enzyme activity. The addition of lymphokine made no significant difference to these figures.

One of the problems with using tetrazolium salts in cytochemical reactions is that they can be nonspecifically absorbed into cells and then be reduced. This may be reflected in high control values. The level of this “activity” was determined in normal cultured macrophages and macrophages treated with control or lymphokine preparations. Figure 1 shows that NBT gave higher background readings than MTT when incubated with normal macrophages. The variation from one specimen to another was very small and no significant difference in this tetrazolium absorption was seen when PMS was added in the incubation medium. Succ. D has a specific competitive inhibitor, sodium malonate. Figure 1 also shows that this inhibitor blocked the activity for Succ. D completely leaving only the level of tetrazolium reduction shown to occur nonspecifically. 7’he Ejfect

of d-Day

Culture

Tt was previously reported that a considerable fluctuation of enzyme activity occurred in the macrophages during the first few days of culture (6). This appeared to stabilise after 6 days and to remain relatively constant up to 14 days. Using the quantitative techniques described above, an initial comparison was therefore made between the activity of the macrophages, just seeded onto the coverslips (2 hr culture), and the activity after 6 days of culture. There was no significant difference in the activity of Succ. D, NADPH oxidation or overall G-6-P.D. (+ PMS) activity. The G-6-P.D. activity, demonstrable without PMS, was slightly raised after 6 days of culture (Fig. 2). As subsequent work (21) involved 3 days of culture with lymphokine, a 6-day period of normal culture was used to allow the cells to stabilise.

18

POULTER

AND

TURK

P>0.1

f

1

PBO.1

100

2 2z 80 fi x = m = E

60 LO 20

MTT NBT 0i

G-6- P.D.

G-6-P.D.+

1

MTT

PMS

FIG. 3. G-6-P.D. activity in normal cultured macrophagesdemonstrated with and without PMS using either NBT or MTT as the hydrogen acceptor. The calculated percent hydrogen available for biosynthesis detectedwith both acceptorsis also shown (mean * SE).

The Use of Different

Tetraxoliuvvt

Salts

MTT and NBT are both rapidly reduced to insoluble coloured formazans, have a similar electropotential (Eo’ Mv+ I .50- + 110) and are both insensitive to oxygen (22). As NBT is a di-tetrazole and is approximately twice the molecular size of MTT, it requires two molecules of hydrogen to reduce it whereas MTT only requires one. This difference in reduc,tion equivalents needed for formazan production is taken into account in the calculation (see Materials and Methods). To examine whether similar results could be obtained with these different hydrogen acceptors, G-6-P.D. activity was assayed in normal macrophages cultured for 6 days using both ‘these tetrazolium salts. It was found (Fig. 3) that whichever tetrazole was used, the G-6-P.D. activity was the same, with or without the addition of PMS, and the extrapolated figures representing the proportion of hydrogen available for biosynthetic purposes was also the same. After 1-hr culture with lymphokine there appeared to be a decreased G-6-P.D. activity in the macrophages when NBT was used as the hydrogen acceptor (Fig. 4A). This decrease was significant in relation to the overall enzyme activity (using PMS) but not significant in relation to that hydrogen picked up by the tetrazole alone. When the same experiment was repeated using MTT as the hydrogen acceptor (Fig. 4B) the apparent &crease in overall G-6-P.D. activity recorded using NBT was abolished and the level of hydrogen picked up by the MTT alone was raised after 1 hr contact with lymphokine. The demonstrable level of G-6-P.D. activity in the macrophages exposed to the control fractions was the same whether NBT or MTT was used in the cytochemical test. The Eflect

of Lynzphokine

1. In C&wed

hydrogen

on Respiratory

Macrophages.

acceptor, 1-hr culture

Activity

It can be seen (Fig. 4B) that using MTT as the with lymphokine had no effect on the overall

K.\PID

EFFECTS

OF LYBI PHOKINE

OS

\I .\(‘ROl’ll

.\GE.i;

1 (I

PC005

r G-6-PO -~

__---I

G-6-P

G-6%PD * PMS

‘-

3 T

G-6-P; .PM??,

MIT

NBT

FIG. 4. The effect of I-hr contact with lymphokine or control G-6-P.D. activity demonstrated \vith: (A). NBT a? the hydrogen the hydrogen acceptor. Values are mean k SE.

fractions acceptor;

on nlauophage (131 &ITT as

G-6-P.D. activity but did increase the activity demonstrated 7~ifhozrf the use of PMS. Because of this a net decrease in the amount of hydrogen available for biosynthesis was recorded (Table 2). KADPH.0 and Succ. I) activity were al~+o measured after l-hr contact with the lymphokine. NADPH oxidation was incrrasctl at this time (Fig. S), but no effect on Succ. D activity was detected. 2. In liver scctio~. Lymphokine or control fractions were added to the cytochemical reaction medium at the same concentration as tlsetl in the macrnllhagt cultures. These reaction mixtures were then used to demonstrate respirator!’ ‘q liver. It was fount1 that enzyme activity in cryostat sections of normal guinea plb the presence of lymphokine did not affect the NADI’IH oxidation or the o\-era11 G-6-P.D. activity (Fig. 6). However, the level of hydrogen produced by G-6-l’.l>. dehydrogenase passing along the endogenous KADPH oxidation pathway ant1 picked up using the tetrazolium salt alone was increased in the prcscncv of lymphokine. By extrapolation a net decrease in the hydrogen available for l)ic)synthesis was seen (Table 3). The Effect

of Lywaphokinr

OH Lysosolrzal

dctizd?

After 1-hr culture with lymphokine or control fractions, the p-glilclirotli(l;Ls( activity of the macrophages was examined. No significant change in activiti was detected (Fig. 7) although a wide \-ariation of activity from cell to cell W;L sre11 both in control and lymphokine-treated cultures, as is demonstrated 1,~ plotting the population histogram.

After 6 days of culture very little SS protein could be detected in the macrol)hages (Table 4) but after 1 hr contact with lymphokine the proportion of SS l)t-otein rose dramatically. This rise was qparently due to the oxidation of S 1-I groups as no increase in the overall concentration of sulphur containing protein was cletectable. Although the control fraction also caused an increase in the proportion of SS groups this remained significantly lower than the !evel recorded after contact \vith lymphokine. As controls were performed using :\‘-ethyl maleimide i 17) results of

20

POULTER

AND TURK

TABLE

2

EXTRAPOLATED PERCENT HYDROGEN AVAILABLE FOR BIOSYNTHESIS Regimen

Addition

6 Days normal culture +l hr culture with test fractions

(G-6-P.D. + PMS) - (G-6-P.D.) (G-6-P.D. + PMS)

No addition

52

Control

58

fraction

Lymphokine”

0 Final concen&ration

equivalent

a

33

to that which inhibits

nonspecific reduction of ferricyanide from the results.

x 100~

macrophage

(which were minimal)

migration

by 50%.

have been subtracted

DISCUSSION Two of the functional properties of lymphokine are macrophage migration inhibition (1, 2) and macrophage activation (3, 4, 6). The factors responsible for these effects are indistinguishable in terms of their Sephadex G-100 elution patterns, buoyant density, and sensitivity to neuraminidase (4). It has been suggested that the changes that occur rapidly after contact with lymphokine render the cells receptive to ac,tivation at a later stage (4). If migration inhibition and activation are mediated by the same factor the question arises as to whether there is a direct relationship between these two phenomena nor whether they are separate effects of the same or very similar substances. It seems likely that migration inhibition is a functional manifestation of cytochemical changes on or within the cell. Because of this and the fact that increases in cytochemical activity have been shown to be characteristic of activated macrophages (3,4,6), we have attempted in the present paper and subsequent work (21) to use various cytochemical techniques to try and rationalize the apparently paradoxical effects of this mediator.

TABLE

3

EXTRAPOLATED PERCENT HYDROGEN AVAILABLE FOR BIOSYNTHESIS Addition

Regimen

Frozen section guinea pig liver

a Final concentration

(G-6-P.D. + PMS) - (G-6-P.D.) (G-6-P.D. + PMS)

No addition

74

Control fraction added to reaction medium Lymphokine* added to reaction medium

82

equivalent

to that which inhibits

67

macrophage

migration

by 50%.

x loon

0

KXPIDEFFECTSOFLYMPHOKINEON PC005

21

MA\(‘KOPHAGES

q Control lractlon q Lymphokino P>O.I

NADPH 0 10’ incubation FIG. 5. NADPH.0 lymphokine or control

Suer D 20’ incubalion

and Succ. D activity in cultured fractions (mean 2 SE).

macrophages,

after

1 hr contact

with

Although there is a significant loss of cells by leaving the macrophages in normal culture for 6 days before the addition of the test fractions, a stable situation is reached in those cells remaining where the effects of lymphokine can be assessed without being influenced by changes due to maturation and differentiation of the exudate cells (6). It is also important when using cytochemical tests on whole cells to try and ensure that the reactants in the incubation medium can enter the cells. The fact that the chemicals actually enter the cells can be demonstrated in a variety of ways. In the present study the demonstration of succinate dehydrogenase activity which is completely inhibited by the specific inhibitor sodium malonate suggests that the chemicals are in contact with the mitochondrial membrane to which Succ. D is bound and must therefore be in the cell. Also the demonstration here of P-glucuronidase activity and, previously (16)) the demonstration of acid phosphatase activity in a particulate form indicate that the reactants can enter even the lysosomes. Seligman and Plapinger (23) demonstrated in an electron microscope study that one could obtain intramitochondrial localisation of tetrazolium salts. Diengdoh and Turk (24) described intracellular localisation of reaction products in macrophages spun onto slides with a cytocentrifuge. These observations were further substantiated by the fact that placing the cell spreads in a solution of histamine, a substance known to increase cellular permeability,

P>OI

G-6-P D

G-6-P.0 + PMS

NADPH 0

FIG. 6. Respiratory activity in frozen sections of normal guinea pig liver demonstrated with lymphokine (shaded) or control fractions added to the inc1~0utio~~ wrdim~~. G-6-P.D. c PMS, IO-min incubation; NADPH.0. 5 min incubation; mean t SE.

22

POULTER

1 LO,

+Lymphokine

‘,cn

AND

TURK

fraction

;‘-,

21-30 40 50 60 70 80 90 100 110 120 absorption Relative

Frc. 7. p-Glucuronidase activity in cultured macrophages after 1-hr contact with lymphokine or control fractions. The relative absorption readings are presented as a population histogram to demonstrate the wide variation of activity from cell to cell. The population of total readings that fell within each arbitrary range of relative absorption is expressed. Mean f SE is also given. increased the demonstrable enzyme activity. One possible reason for the apparent decrease in enzyme activity reported to occur 1 hr after lymphokine contact (6) is that the lymphokine reduced the permeability of ‘the cell membrane. Using the elution technique for quantitating the demonstrable enzyme activity it has been possible in the present study to compare the use of a ditetrazolium hydrogen acceptor to a mono-tetrazole. It was found that, using the small molecular weight tetrazole (MTT), the apparent decrease in enzyme activity ,that appeared to occur after lymphokine contact was not demonstrable. As the levels of activity in the macrophages exposed to control fractions were the same whichever tetrazolium salt was used, the differing results, after lymphokine contact, were taken to imply that a decrease in cellular permeability was occurring and this was adversely affecting the entry of the di-tetrazole molecule into the macrophage. These two tetrazolium salts do differ in charge and in other radicals on the molecules both of which could possibly affect their ability to enter the cell. As the macrophages being studied are “dead” however (having been allowed to dry), the most likely explanation for the lower activity using NBT is one of mechanical hindrance to molecular entry. It must also be remembered that these cells (being dried) may not reflect the membrane conditions of the living macrophages. It is suggested TABLE 4 -SS AND

-SH

PROTEIN

RATIO

AFTER

1 HR CONTACT

WITH

LYMPHOKINE

Conditions

-SH

TotaP

ss (%I

N. culture $-Control sup. +Lymphokine

29 f 2b 30 f 4

30 f 1 47 f 3

3 36

18 f

4.5 f 5

60

2=

a After 30-min treatment with 20$&sodium dithionite at 37’C. bMean relative absorption f SE. c P < 0.02 when comparedwith control.

RAPID

EFFECTS

OF

LY

MI’IlOKIKt?

ON

1.3

MA(‘KOl’ITACES

however that changes to the state of the cell membrane that have occurred when the cells were alive might be the cause of the apparent differences in characteristics seen in the specimens studied. As it is very difficult to retain enough cells 011 the coverslips of “wet” preparations to quantiate their activity it is not known whether changes in activity (if they occur) due to drying are different in cells treated with lymphokine. It seems difficult to imagine however that these artefacts are responsible for totally differing results when one compares the results in this paper to those occurring in cells after longer contact with lymphokines (21 i. Changes in the proportion of SH and SS groups in cell protein add weight to the suggestion of decreased permeability following lymphokine contact, as the relative proportion of these groups has been shown to affect membrane intrgrit? (25).

It

is

alsO

inter&hlg

t0

note

that

this

effeCt

CJf ~yniph0kille

ilf

‘i’ifro

C0rreht(‘S

with the decreased permeability of peritoneal macrophages in Go when sensitizrcl animals are injected intraperitoneally with antigen ( 21) Using MTT as the hydrogen acceptor the overall GG-P.D. activity was unaffected by l-hr contact with lymphokine while the endogenous NADPH oxidation fueled by G-6-P.D. activity was raised. It would appear therefore that the lytnphokine altered the way the cell utilised the hydrogen liberated by the G-G-P.D., increasing N4DPH oxidation at the expense of biosynthesis. The SADPH oxidation system is an essential component of the peroxidase-halide bactericidal mechanism (26) as it can produce hydrogen peroxide. \Vhen the activity of this system was measured independently using KADPI-I as a substrate, this was also seen to be raised after lymphokine contact. However. no increase of Succ. 1) activity could be detected after 1-hr culture with lyniphokine, suggesting that there was a certain selectivity in this rapid response and not all respirators pathwa\-s \vere affected. In relation to lysosomal enzyme activity, no effect of lymphokine on ,&glucuronidase activity was seen. This result agrees with earlier observations of acid phosphatase activity which was also unaffected b)- l-hr lymphokine contact (6). As the demonstration of lysosomal enzyme activity cytocheniically is determined in part b\ the integrity of the lipoprotein membrane of the lysosome (27, 28) it is not surprising that no increases in activitv were seen at a time \\.lien membrane pcrin~ability appears to be decreased. It is still unclear however whether the membrane changes are ;I direct effect of the lymphokine or a result of the intracellular changes. Decreases in DNA and RNA synthesis have already been reported as effects of Iymphokine containing supernatant fluids, using Change liver cells (29). L4lth0ugll the time course of these changes was longer than 1 hr, the unfractionated, niitogei:-intlucetl fractions used in that work may contain other factors missing from the lymphokine employed in the present study. In conclusion it is suggested that 1-hr culture with lymphokine can directly affect HhlPS activity in macrophages and that these changes. primarily in utilisation of the liberated hytlrogen, result in increased NADPH oxidation at the expellse of biosynthesis. ~111overall decrease in cellular permeability also occurs, but it is postulated that this is a result of changes occurring Lvithin the cell. The increase in N.qI)PH oxidation may be a rapid attempt to increase the bactericidal capacity of the cell through the peroxidase-halide system which has recently been postulated to reside in macrophages (30’1. In any case this system is kno\f~l to occllr in l~olyn~orl)l~o~~~~cle;~r

24

POULTER

AND

TURK

leukocytes (22) and peritoneal exudate monocytes (31) and there is no reason to assume that the macrophage is the only cell that could be affected by these factors. This hypothesis will be discussed in more depth after subsequent work (21).

ACKNOWLEDGMENT The authors thank Miss Lynn Norman for her expert technical assistance.

REFERENCES 1. 2. 3. 4. 5.

Bloom, B. R., Advan. Zmmunol. 13, 101, 1971. David, J. R., N. Eng. J. Med. 288, 143, 1973. Nathan, C. F., Karnovsky, M. L., and David, J. R., 1. Exp. Med. 133, 1356, 1971. Nathan, C. F., Remold, H. G., and David, J. R., J. Exp. Med. 137, 275, 1973. Dumonde, D. C., Wolstencroft, R. A., Panayi, G. S., Matther, M., Morley, J., and Howson, W. T., Nature (London) 224, 38, 1969. 6. Nath, I., Poulter, L. W., and Turk, J. L., Clin. Exp. Immunol. 13, 455, 1973. 7. Brogan, T. D., In “The Reticuloendothelial System and Immune Phenomena” (N. R. Di Luzio and K. Fleming, Eds.), p. 267. Plenum Press, New York, London, 1971. 8. Poulter, L. W., and Turk, J. L., Cl& Exp. Znznmnol., in press. 9. Wolstencroft, R. A., and Dumonde, D. C. In “Zn Vitro Methods in Cell-Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.) Academic Press, New York, London, 1971. 10. Chayen, J., Bitensky, L., Butcher, R. G., and Poulter, L. W., “A Practical Guide to Histochemistry,” Oliver and Boyd, Edinburgh, 1969. 11. Shelton E., and Rice, M. E., J. Nat. Cancer Inst. 18, 117, 1957. 12. Altman, F. P., Biochenz. J. 114, 13P, 1969. 13. Glock, G. E., and McLean, P., Biochem. J. 55, 400, 1953. 14. Altman, F. P., Progr. Histochem. Cytochem. 4, No. 4, 1972. 15. Wulff, H. R., Acta Haematol. 32, 17, 1964. 16. ChCvremont, M., and Frederich, J., Arch Biol. 54, 589, 1943. 17. Casselman, W. G. B., “Histochemical Technique.” Methuen, London, 1959. 18. Butcher, R. G., Histochemie 13, 263, 1968. 19. Chayen, J., and Denby, E. E., “Biophysical Technique.” Methuen, London, 1968. 20. Poulter, L. W., M. Phil. thesis. University of London, 1972. 21. Poulter, L. W., and Turk, J. L., Cell Immunol. 20, 25-32, 1975. 22. Altman, F. P., Histochemie 22, 256, 1970. 23. Seligman, A. M., Nir, I., and Plapinger, R. E., J. Histochem. Cytochem. 19, 273, 1971. 24. Diengdoh, J. V., and Turk, J. L., Znt. Arch. Allergy 31, 261, 1967. 25. Chayen, J., Bitensky, L., Butcher, R. G., and Poulter, L. W., Nature (London) 222, 281, 1969. 26. Klebenoff, S. J., J. Exp. Med. 126, 1063, 1967. 27. Chayen, J., and Bitensky, L., In “The Biological Basis of Medicine” (E. Bitter and N. Bitter, Eds.), p. 349, Academic Press, London, New York, 1968. 28. Chayen, J., Bitensky, L., Butcher, R. G., Poulter, L. W., and Ubhi, G. S., Brit. J. Dermatol. 82 (Suppl. 6), 97, 1970. 29. Polet, H., Cell. Immunol. 11, 247, 1974. 30. Romeo, D., Cramer, R., Marzi, T., Saranzo, M. R., Zabucchi, G., and Rossi, F., J. Reticuloendothelial Sot. 13, 399, 1973. 31. Daems, W. T., and Brederoc, P., In “The Reticuloendothelial System and Immune Phenomena” (N. R. Di Luzio and K. Fleming, Eds.), p. 19, Plenum Press, London.