Lipids and lymphocyte function

Immunology Today, rot. 5, No. 3, 1984

70 14 Waldschmidt, T., Borel, Y. and Vitetta, E. (1983)J. ImmunoL 131, 2204-2209 15 Metcalf, E. S. and Ktinman, N. R. (1976)J. Exp. ivied. 143, 1327-1340 16 Pike, B. L., Boyd, A. W. and Nossal, G . J . V . (1982) Proc. NatlAcad. Sci. USA 79, 2013-2017 17 Venkataraman, M. and Scott, D. W. (1977)J. IramunoL 119, 1879-1881 18 Benjamin, D. C. (1975)J. Exp. Med. 141,635-646 19 Basten, A., Miller J. F. A. P., Sprent, J. and Cheers, C. (1974)J. Exp. Med. 140, 199-217 20 Taniguchi, T. and Miller, J. F. A. P. (1977),]. Exp. Med. 146, 1450-1453 21 Parks, D. E., Doyle, M. V. and Weigle, W. O. (1978)J. Exp. Med. 148,

625-638 22 Tada, T., Okuraura, J~. and Taniguchi, M. (1973)J. lmmunol, t11, 952-961 23 Gershon, R. K. and Kondo, K. (1971) Immunology 21, 903-914 24 Scott, D. W., Long, C.,Jandinski, J . J . and Li, J. T. C. (1980) Immunol. Rev. 50, 275-309 25 Nemazee, D. A. and Sato, V. L. (1983)J. Exp. Med. 158, 529-545 26 Bona, C., Finley, S., Waters, S. and Kunkel, H. G. (1983)J. Exp. Med. 157, 986-999 27 Triplett, E. L. (1962)J. Immunol. 89, 505-510 28 Rollins-Smith, L. and Cohen, N. (1982) Nature (London) 299, 820-821

Lipids and lymphocyte function K, N. TraiU and G. Wick Lipids have a variety of important biological functions, serving as storage and transport forms of metabolic fuel, as structural membrane components, and as cell-suorace components conferring, for example, tissue and species specificity. Not surprisingly, because the immune system functions through a complex series of stimulatory and regulatory interactions, there has been speculation about the role of lipids as lymphocyte membrane components. Here Kay TraiU and George Wick discuss recent research on the possible influence of lipids on receptor binding, signal transmission and the stimulation of lymphocytes to effectorfunction.

Membranes: structure, fluidity and signal transmission

Structure andfluidity The fluid-mosaic model of membrane structure pro posed by Singer and Nicolson in 19722 still encompasses most of the known features of membrane structure and function. Membranes are composed primarily of lipids and proteins; the lipids serve the dual purpose of forming a permeability barrier to ions and (most) polar molecules and providing a fluid solvent for membrane proteins; while the proteins perform the membrane functions, such as transport, communication and energy transduction. The proportions of lipid and protein vary depending on the source of the membrane but active plasma membranes, such as those of the lymphocyte, contain many pumps, gates, receptors and enzymes and have a typical protein content of about 50%. The lipid bilayer itself is generally highly asymmetrical in terms of the lipid composition of the outer and inner layers. The molecules diffuse freely in the plane of each layer, but a 'flip-flop' across the membrane from the outer to the inner bilayer (or vice versa) is a very rare event. Integral protein molecules are usually also asymmetrically oriented and may be exposed on one or the other surface or may span the whole membrane. The latter, as well as those exposed only on the inner surface of the membrane, may be associated with the cytoskeleton. While such proteins may be relatively immobile, most are predicted to float free in the lipid 'sea', their motion retarded only by the viscosity of the lipids and by protein-protein collisions and interactions. Comparison of lateral mobilities of lipids and proteins using the technique of fluorescence recovery after photobleaching (FPR) has shown that lipid diffusion coefficients are constant compared with those of proteins (approximately 10-8 cm 2 s-1) and generally at least an Institute for General and Experimental Pathology, University of Innsbruck Medical School, A-6020 Innsbruck, Austria. © 1984,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/84/$02.00

order of magnitude greater; diffusion coefficients for proteins are very variable (ranging from 4 x 10-0 to 10-12 cm 2 s- 1) and a certain proportion of any protein fraction studied is usually immobile in the plane of the membrane 3. The !;p;d fll~id~ty i~ nhvlnn~lv not the major retarding factor of this type of protein lateral mobility but it may play an important role on a much shorter distance scale, influencing the rotational diffusion of protein molecules and their aggregation after cross-linking3; the conditions (lipid composition) immediately adjacent to a particular protein (enzyme or receptor) under study are therefore of particular relevance. Recent evidence has indicated that even at physiological temperatures membrane lipids segregate into 'domains' or discrete regions in the liquid crystal state adjacent to regions in the gel state 4'~ (Fig. 1). Certain proteins have been shown to partition preferentially into one or other type of domain; thus the local environment of a protein may be expected to remain constant despite gross changes in membrane composition and in these situations a poor correlation would be expected between altered membrane lipid composition and function of this protein. The existence of lipid domains necessitates questioning the significance of the popular concept of an 'optimal fluidity' of membrane lipids for 'optimal performance' of physiological function. Nevertheless, homeostatic regulation of membrane lipid composition is extremely strict and the observation that cells grown in a medium lacking cholesterol compensate by increasing the incorporation of saturated fatty acids 6'7 lends support to the concept that maintenance of 'fluidity' is more relevant than actual membrane composition. Lipid fluidity is decreased by increasing the ratios of saturated/unsaturated fatty acids, sphingomyelin/lecithin and cholesterol/phospholipids. Cholesterol acts by insertion into the membrane bilayer between the phospholipid chains in such a way that it abolishes the phase transitions of the fatty acids: below the phase-transition temperature it prevents the formation of the rigid gel

7t

ImmunologyToday, voL 5, No. 3, 1984

alterations in the plasma m e m b r a n e C/P1 of chicken PBL arising from in-vitro modulation (see next section).

~CHO

~

jperipherQI

Fig. 1. Membrane model depicting fluid and gel (or less fluid) phospholipid domains with integral proteins 'a' and 'b' situated in the fluid domain and integral protein 'c' in the gel domain. The different degree of partitioning of cholesterol is schematically depicted. phase by disturbing the packing of the phospholipid chains, and above the phase-transition temperature it reduces the mobility of the chains. T h u s at physiological temperatures an increase in the cholesterol/phospholipids ratio (C/P1) of the m e m b r a n e reduces its 'fluidity'. 1n-vitro modulation of m e m b r a n e cholesterol content has been used as a n experimental approach by m a n y investigators to study the effect of altered 'fluidity' on m e m b r a n e protein lateral and rotational mobility, vertical position a n d m e m b r a n e function. Fluidity can be assessed in a n u m b e r of ways (reviewed in Ref. 3) but the most c o m m o n (and perhaps therefore the most controversial) method has been that of measurement of the depolarization of hydrophobic probe molecules such as D P H (1,6-diphenyl 1,3,5-hexatriene), the measurements providing an assessment of the environmental constraints upon the rotation of the probe molecule inserted between the acyl chains of the m e m b r a n e phospholipids. Criticisms of the technique are manifold, ranging from objections to the use of probe molecules which m a y perturb the m e m b r a n e (but are also required for other techniques, for example electron spin resonance and FPR), and reservations about the meaningfulness of 'average' fluidity measurements especially when a probe may partition differently into different domains or exhibit different excited lifetimes in different environments, to objections to D P H itself which to a certain degree partitions into internal m e m b r a n e s (i.e. of various cell organelles) as well, thereby giving a resultant m e a n of the fluidity of all the cell m e m b r a n e s 4's. It is generally agreed that D P H polarization measurements can be used as an indication of change in lipid composition of the m e m b r a n e although extrapolation to 'microviscosity' expressed in poise may be presumptive. Fig. 2 depicts two examples of the use of D P H in chicken lymphocytes, the chicken being of special interest to us as an experimental model for ageing and a u t o i m m u n e disease 9'~°. In the experiment shown in Fig. 2A it was used to show differences between thymus, spleen and peripheral blood lymphocytes (PBL) of chickens, the thymus cells (immature) showing lower values (more fluid membranes?) than mature cells, as predicted s. In the experiment shown in Fig. 2B it was used to monitor

Signal transmission Signal transmission to a cell requires b i n d i n g and crosslinking (aggregation) of cell receptor molecules by the respective triggering hormone, lectin, antigen, i m m u n o globulin (Ig), etc. The evidence that hormone function can be mimicked by bivalent F(ab)2 anti-receptor antibodies but not by the monovalent Fab has lent sup= port to the physiological importance of the cross-linking step4'~L Compositional changes in the local lipid environment of a receptor molecule m a y be expected to affect both binding and cross-linking events, evidence for which will be discussed in the next section. Vertical packing or positioning of surface molecules can also be affected by lipid composition s . Patched receptors rapidly become immobilized a n d are then drawn into a cap through interaction with the cytoA

B

~ 0.30" +1

g 0.28. -'tO. Q

"5 0.26g 1 ~0.22 a 0.20

/ T h y Spl PBL

controlcholesterolAL rug/ml

- /aglml

Fig. 2. (A) Differences in the fluidity (composition) of thymus (Thy), spleen (Spl) and peripheral blood lymphoeytes (PBL) cell membranes demonstrated using the fluorescence depolarization technique with the probe 1,6-diphenyl 1,3,5 -hexatriene (DPH). Washed cellswere labelled with DPH by incubatingthe cells (5 x 106 m1-1) with a DPH suspension (2 x 10 -6 M in phosphatebuffered saline) 1/1 (vol./vol.) for 30 min at room temperature. The degree of polarization 'p' of DPH was measured at 25°C as described in Ref. 49. Triplicate measurements were made on cell suspensions from each individual chicken; results are expressed as mean -+SD of 'p' for six 3-week-oldchickens. • p < 0.05 compared with PBL. • • • p< 0.001 compared with PBL. (B) Differences in the degree of polarization 'p' of DPH after modulation of PBL membrane lipid composition. PBL (5 x 106 ml-1) from four adult chickens were incubated (separately) overnight at 37oc in Iscove's medium containing0.5% bovine serum albumin, BSA) enriched with: 50 or 100/agcholesterol ml-i; 125 or 500/ag 'active lipid' mixture (AL) ml-1; 1% ethanol (control). On the next day they were washed 3 times in phosphate-buffered saline and resuspended at 5 × 10s ml - l for incubationwith DPH, as in (A); measurements shown were performed at 41°C (body temperature of chickens) because the effect of cholesterol was less pronounced at lower temperatures. •• • p< p < 0.05 0.01 I • • • p< 0.001

compared with control.

72

Immunology Today, vol. 5, No. 3, 1984

TABLE I. Phospholipidmethylationand signaltransductionin a varietyof cell types" Cell type

Stimulus

R a t reticulocyte C 6 glioma a s t r o c y t o m a

fl-Adrenergic agonists /3-Adrenergic agonists, benzodiazepine agnnists O-Adrenergic agonists C o n A IgE receptor IgE-specific antigens Con A Chemotactic peptides Bradykinin T h r o m b i n , adrenaline

H e L a cells M a s t cells Leukemic basophils Lymphocytes Neutrophils Fibroblasts Platelets

Phospholipid methylation + + + + + + + + No effect

Biological effect Cyclic A M P Cyclic A M P ? Cyclic A M P C a 2÷ influx, histamine release H i s t a m i n e release C a 2+ influx, mitogenesis Chemotaxis Cyclic A M P Aggregation

aAdapted from ReL 13.

skeleton. This may require association of the receptorligand complex with a cytoskeleton-attached binding protein. Cis unsaturated fatty acids inhibit capping of membrane Ig, perportedly by partitioning into the membrane in regions surrounding Ca2+-carrying proteins and thereby altering lipid-protein interactions causing release of Ca 2+ and disrupting cytoskeleton interactions ~2. Within minutes of binding and aggregation of receptor molecules many membrane enzymes and ion channels become activated. Among the very early events, even preceding Ca 2÷ influx, is the activation of two methyl transferase enzymes which act to methylate phosphatidylethanolamine (PE) to phosphatidylcholine (PC) and cause its transfer to the outer side of the bilayer with a resultant increase in membrane fluidity. This reaction has been closely correlated with Ca 2+ influx, cyclic A M P generation, phospholipase A activity, and effector function of the particular cell ~3(Table I). Another early event after triggering is the activation of the acyl CoA lysolecithin acyltransferase enzymes 14which increase the incorporation of unsaturated fatty acids into phospholipids. These enzymes are spatially associated with the highaffinity concanavalin A (Con A) receptor on rabbit lymphocytes and it is proposed that their activation may cause

a wave of phospholipid fluidity increase along the membrane, thereby propagating the induction signal and modifying (activating) many other membrane enzymes. Activation of both of these groups of enzymes would account for the rapid increase in fluidity of the lymphocyte membrane shortly after stimulation (within 30 rain) which returns to normal within 1 h ~5'16. These rapid initial events are followed by sequential synthesis of lipids and steroids, R N A and proteins, and DNA. In-vitro modulation of the lipid composition of

lymphocyte plasma membranes Methods

The cholesterol and phospholipid content of membranes can be modulated by incubation of the cells with cholesterol/phospholipid liposomes of varying mole ratio (for example, see Table II), when cholesterol freely equilibrates between the liposome and cell membrane. However, it should not be forgotten that liposomes may also fuse with the cell membrane such that, for example, pure lecithin liposomes reduce the plasma membrane C/P1 both by extraction of cholesterol and by fusion and enrichment with lecithin. An additional complication

T A B L E II. Effect of lipids on mitogen response of lymphocytes Species

Lipids

Pre-incubation

Enrichment/depletion tested by

Serum supplement

Mouse a Mouse a Mouse a

Liposomes Liposomes Liposomes

1.5-2 h, washed 4-24 h 15-20 h

DPH polarization Membrane cholesterol D P H polarization

Chicken t'

Ethanolic 24 h, washed solutione Ethanolic 24 h solutione Ethanolic None solutione

D P H polarization

Chicken b Chicken b Bovinec Human b Human b Human b

Liposomes Liposomes Liposomes f Liposomes

16 h 24 h None 2 h, washed

Effect on mitogen C/P1 increase

Response of C/PI decrease

Refs

FCS ? FCS lipoprotein depleted

~ ~ ~

~ --~

23 22 24

None

~

±

DPH polarization

None

-

~

Not tested

None

~

~

K . N . Traill and G. Wick, unpublished observations K . N . Traill and G. Wick, unpublished observations K . N . Traill and G. Wick, unpublished observations

Membrane cholesterol Membrane cholesterol Not tested Not tested

FCS FCS FCS FCS

~ ~ ND ND

ND ~ ~ "~

20 21 25 25

Abbreviations: ND, not done; FCS, fetal calf serum; DPH, 1,6-diphenyl 1,3,5-hexatriene; C/PI, cholesterol/phospholipids ratio. a Spleen cells. b Peripheral blood lymphocytes. c Lymph node cells. d Mitogens most commonly used have been concanavalin A (Con A) and phytohemagglutin (PHA) but others have also been included. e Culture performed in Iseove's medium 36 modified for chicken cells, supplemented with 0.5% bovine serum albumin (BSA) and enriched with 100 gg cholesterol m l - 1 or 500/ag 'active lipid' mixture (AL) ml - 1 . f Suppression observed only at higher concentrations (300/ag ml - 1); 60 gg ml - I did not suppress the PHA response and enhanced the Con A response.

Immunology Today, vol. 5, No. 3, 1984

arises through the possibility of adherence of the liposomes to the cell surface (i.e. without fusion) which would lead to 'false' results for fluidity measurements and membrane composition analysis. Simple dispersions of cholesterol or its hydrophilic esters (for example cholesterol hemisuccinate, CHS) can be prepared in culture medium by dilution (rapid injection) of ethanolic stock solutions into the medium, and these can be successfully used for modulation of the membrane C/P1 (Ref. 17). Some people have found that cholesterol supplied to cells in this way is not readily available for uptake t8 but we (Fig. 2) and others ~9find that at least some is taken up. Particularly efficient for fluidization of membranes is the 'active lipid' mixture (AL)* which we use (Fig. 2); it is a mixture of neutral glycerides, lecithin and phosphatidyl ethanolamine which forms micelle structures when dispersed in water, the neutral lipids forming the core and the phospholipids distributed on the surface a. Incubation times used by different groups for enrichment of cholesterol and phospholipids have varied from 1.5 to 24 h (Table II). This probably reflects experimental convenience as well as technical differences and differences in the lymphocyte source. Shintzky et al. prefer to work with CHS because its incorporation is much more rapid than that of cholestero117. In a 3-h incubation they find significant changes with C H S but not with cholesterol, which is in keeping with our observations that an overnight inc!:hation (ie !ongor than g h] nfchleken PBL with cholesterol is required to demonstrate significant differences in the degree of polarization of DPH. Monitoring of C/P1 differences has been performed both by analyses of membrane lipids z°~2~ and by assessing differences in membrane fluidity (Refs 23-25; K. N. Traill and G. Wick, unpublished observations).

Effect of modulation on antigen (receptor) expression and mobility Shinitzky and co-workers have proposed a model whereby changes in the C/P1 and microviscosity of a membrane would cause alterations in the lipid freevolume and in the solubilization capacity such that membrane proteins would be displaced from their equilibrium position 'vertically' or 'laterally' to a new equilibrium position (for review, see Ref. 8). They demonstrated by spectral and chemical methods, as well as by ligand binding, that certain erythrocyte antigens (for example Rh ' D ' antigen) become vertically displaced (more exposed) with increasing membrane C/P1 and vice v e r s a 8'26'27. Recently they have reported that the Thy-1 antigen on EL4 cells behaves in the same way, demonstrated simply by measurement of intensity of fluorescence staining after incubating modulated cells with fluorescein-labelled anti-Thy antisera. Their model predicts that different membrane proteins would (or could) be affected differently depending on their position in the membrane, and they have preliminary data s that mouse H-2 antigens may become less exposed with increasing C/P1. It may be that this type of modulation of receptor sites has physiological significance in terms of immune regulation since the authors have shown that monokines, such as lymphocyte-activating factor, upon interaction *Gift from M. Shinitzky, The Weizmann Institute of Science, Rehovot, Israel.

73

with their receptor on lymphocytes induce microviscosity changes in the lymphocyte plasma membrane while simultaneously inducing an increase in the number of cells binding the polymeric antigen TGAL, perhaps by exposing cryptic receptor sites. Mimicking of the microviscosity increase by increasing the membrane C/P1 also resulted in an increase in antigen-binding cells, leading the authors to predict that the purpose of LAF may be to prepare the T-cell for binding of (H-2-restricted) immunogens 28. Shinitzky and colleagues have elicited strong skin-test reactions to autologous turnout cells by modulation of the tumour cells with CHS, probably also attributable to unmasking of tumour-associated antigens 17. In model membrane systems cholesterol enhances hapten exposure to antibody-binding sites 29 but there appears to be no difference in mitogen (Con A) binding after cholesterol enrichment or depletion of lymphocyte membranes 2°'21, thus altered binding cannot explain effects of cholesterol on the in-vitro mitogen responses reported by these authors (Table II). There are, however, reports of an inverse correlation between the rotational mobility of Con A receptor sites and the fluidity of the plasma membrane 3°, although this was based on a comparison of leukemia cells (high lipid fluidity and low Con A receptor rotational mobility) with normal lymphocytes (low lipid fluidity and high Con A receptor rotational mobility). This rotational mobility of lectin recentors mav be of nhvsiolo~ical imoortance since their mitogenicity is supported by the demonstration that mitogenic lectins are highly mobile after binding to their receptor molecules whereas non-mitogenic lectins are relatively immobile'5'31. Capping is another parameter often used to assess the intrinsic capability of a lymphocyte to respond; for example, cells from older animals/people s h o w a diminished capacity to cap which is reflected in their diminished immune capacity. At least two mechanisms of capping may exist, both of which can be disturbed by alterations in membrane lipids. The first, involving the cytoskeleton, is the energy-dependent movement of patched receptors into the cap which as well as being inhibited by numerous drugs can also be affected by cholesterol or fatty acid perturbation of membrane structures lz'3z. The second type of capping which may exist is cytoskeleton independent and may be explained by a simple accumulation of protein complexes at one pole of the cell as a result of flow of membrane lipids ~3'~4.This type ofcytoskeleton-independent capping of surface Ig on rabbit lymphocytes has been induced by reducing the membrane C/P1 by incubation with pure lecithin liposomes and was prevented by an incremental addition of cholesterol to the liposomes 35. Recovery of surface Ig took approximately 24 h (compared with 6.5 h after anti-Iginduced capping) and was directly dependent upon cholesterol biosynthesis. In contrast, lecithin liposomeinduced capping of Con A receptors was possible only after the cytoskeleton was disrupted which supports the view that the Con A receptor is a transmembrane glycoprotein anchored to the cytoskeleton whereas Ig is not transmembranous and only becomes cytoskeleton-bound after ligand cross-linking.

74 Effect of modulation on in-vitro lymphocytes responses

There have been a number of different experimental approaches to this question and interpretation of the data is generally somewhat ambiguous. Many authors have pre-incubated lymphocytes for varying periods of time with cholesterol/phospholipid liposomes or lipids in solution in order to modulate the membrane lipid composition (fluidity), after which they have stimulated the cells with Con A, phytohemagglutinin (PHA) (or other mitogens) (for example, see Table II). For example, Rivnay et al. 2.~ pre-incubated mouse lymphocytes for 1.5-2 h and washed them before stimulation with Con A in normal tissue culture medium; they found that perturbation of membrane fluidity in either direction (increased or decreased C/P1) suppressed the mitogen responsiveness, providing support for the concept of an optimal fluidity for optimal physiological function. The complication arises, however, that in many experimental designs the lipids/liposomes remain in the culture medium throughout the entire culture period making it difficult to distinguish membrane/stimulatory effects from other cell culture effects. Our own data with chicken PBL clearly illustrate the effect of these technical variations on the outcome of these experiments. PBL pretreated with 100 lag cholesterol ml -~, washed and stimulated with P H A in normal tissue culture medium gave an enhanced response, particularly early in culture. In contrast, stimulation of chicken PBL in cholesterolcontaining medium (100 lag ml -~) without prior incubation always resulted in a slight but statistically significant suppression. Pre-incubation with cholesterol combined with stimulation in cholesterol-containing medium sometimes enhanced and sometimes suppressed, perhaps due to a combination of the two effects. AL, in contrast, only enhanced the P H A response when present throughout the culture period and the effects were most marked late (or later) in culture. Preincubation with AL for 24 h (which resulted in a pronounced increase in fluidity) before culture in normal medium did not affect the P H A response. In addition, when cells were cultured in medium containing an enhancing concentration of AL and a suppressive concentration of cholesterol, they responded as well as, or better than, with AL alone. Lymphocyte stimulation by mitogens needs cholesterol. The cholesterol can be synthesized by the cell itself or can be supplied exogenously in the tissue culture medium, thus a possible effect of cholesterol pre-incubation (other than on the cell membrane C/P1) may be to alleviate the requirement for cholesterol biosynthesis; abrogation of the required cycle of steroid synthesis before DNA synthesis, predictably, results in an earlier response to the mitogen (Ref. 24; K. N. Traill and G. Wick, unpublished observations). It should be noted that most cultures have been performed in 5-15 % fetal calf serum (FCS) - an additional source of exogenous cholesterol (as much as 500 lag free cholesterol ml-l). This is seldom taken into account but can be expected to dampen the effect of any lipid additives: instead serum-free culture conditions can be used (as we have done) or lipoproteindeficient medium 24, The importance of this consideration is demonstrated in a mouse spleen cell culture system

Immunology Today, vol. 5, No. 3, 1984

where 100 #g cholesterol ml - 1 enhanced the response in lipoprotein-depleted FCS but inhibited the response in medium containing normal FCS; 200 lag ml - 1 was inhibitory in both 1~. From appraisal of the results shown in Table II we would conclude: (a) that none of the effects can be unequivocally attributed to altered membrane fluidity and triggering; (b) that there is a requirement for exogenous cholesterol in culture for optimal responsiveness but that excess is suppressive. We cannot predict a mechanism for suppression but cell mortality is clearly not involved; (c) that the suppression by pure phospholipid liposomes reported by others may be explained by depletion of cellular cholesterol and/or there is a requirement for exogenous phospholipids in the culture medium but that an excess of phospholipids is suppressive, again through an unknown mechanism. The enhancement of mitogen responsiveness which we demonstrated with AL (without pre-incubation ) accords with another report on serum-free tissue culture (also performed in Iscove's modified Dulbecco's medium, supplemented with bovine serum albumin, BSA). In this study although a pure soybean lecithin preparation proved inhibitory, an impure mixture of similar composition to our AL (triglycerides, PC, PE, monophosphatidyl inositol, traces of sterol) was enhancing 36. In the system used (mouse splenic B-cell IgM and IgG secretion after lipopolysaccharide, LPS, stimulation) there was little or no cholesterol requirement, perhaps because sufficient cholesterol was supplied in the lipid mixture. Our results indicate that although chicken PBL respond well in Iscove's medium without lipid supplementation (de novo synthesis of lipid?) supplementation with a mixture ot lipids plus cholesterol provides better culture conditions. Recently serum-free (and protein-free) cultures have become popular for short-term (24 h) stimulation to generate growth factors such as interleukin 2 (IL2) 37. Lipids seem to be required not for stimulation but for continued growth in culture. Variable effects (usually suppression) have been reported after supplementation ot culture medium with either saturated or unsaturated fatty acids ~'38. One interesting aspect was demonstrated by Maccachini and Burger who, after including avidin in their culture medium to suppress de novo fatty synthesis and to ensure incorporation of the exogenous fatty acid supplied, found little difference in the Con A response of oleate- and elaidate-substituted cells at 37oc but marked differences when the cells were stimulated at lower temperatures (30-31 oC) 39 Heiniger and Marshall18 have investigated the relative importance of cholesterol synthesis for the polyclonal induction of splenic lymphocytes into cytotoxic cells. They found that there was an absolute requirement for the synthesis of cholesterol which could be counteracted by the inclusion of cholesterol/phospholipid liposomes in the culture medium (apparently the 5% fetal bovine serum in the culture medium was not sufficient) in this instance. DNA synthesis on the other hand was not required. Using a different approach, Dabrowski et al. 40 investigated the role of cholesterol on the membrane of cytotoxic effector cells (antibody-dependent, natural killer and PHA-mediated cytotoxicity) by incubation of

75

Immunology Today, vol. 5, No. 3, 1984

TABLE III. The responseto concanavalinA by peripheral blood lymphocytesfrom donorswith high and low microviscosity,in five different age groups" I

I

I

I

I

I

1.800 f-i

." 1 'o

1.700

1

1.6oo

•

i

o

•

I •

•

1.500

.o 1.400

6 •

0--'

~

•

I

I

20

:

,

~E

'

I

30

" ~

40

Age

•

•

~0

,

i

• •#

'

'

7

;..

tO t ,

•

[

'..

•-%---

..... a

,

.

10

mc

n [

I--

• ,

Microviscosity levelsb High Low

1

I

I

{

I

50

60

70

80

[years]

Effect of modulation of target cells on their susceptibility to lysis Using a panel of drugs to block various metabolic functions, Schlager 41 has investigated the different parameters influencing the susceptibility of target cells to lysis by cytotoxic T cells and by antibody plus complement. The results of this approach confirmed earlier conclusions that the fluidity (i.e. C/P1) of the tumour cell target regulates the cell's susceptibility to complement attack, increased susceptibility correlating with decreased membrane C/P1 and vice versa. In contrast, modulation of susceptibility to cytotoxic T-cell lympholysis was unaffected by changes in the membrane cholesterol/phospholipids mole ratio but correlated with synthesis and content of the highly polar lipids; cells with increased susceptibility to T-cell cytotoxicity had a lower surface charge and vice versa. Lipids or lipid-containing macromolecules in the target cell may also play a role in the outcome oflysis with complement since pre-incubation of complement with complex lipids or fatty acids can suppress or enhance, respectively, its lytic capacity4L Lipoproteins and immune function In addition to the specific immunoregulatory lipoproteins, LDL~N, all normal lipoprotein fractions have been attributed an immunosuppressive role in vitro even when incorporated into culture medium at physiological concentrations 43~47.The physiological significance of this is fascinating but since the regulatory effect is generally receptor mediated and independent of the lipid fraction of the molecule it is beyond the scope of this review. One exception is the experiments of Shine et al. 19on the in-vitro anti-sheep red blood cells (SRBC) plaque-forming cell (PFC) response of mouse lymphocytes cultured in lipoprotein-deficient medium. Low-density lipoprotein-supplemented medium restored immune function and could be partly mimicked by the lipid fraction of low-density lipoproteins and by cholesterol, whereas high-density lipoproteins had no effect.

Conclusions experimental

data

14-24 25-36 37-50 51-65 66-77 211840~ 166861 150803 95505 58641 -+15079 +18125 -+19918 -+19324 +5734 257502 259737 181125 171680 102060 •+12657 -+26406 -+22739 -+29415 -+18998

Reproduced from Ref. 48. b High and low mieroviscosity for each age group refers to the values above and below the dotted lines depicted in the figure. (3H) thymidine uptake, mean c.p.m. -- SE of 4-6 individuals. Each individual sample was measured in quadruplicate cultures.

effector cells with cholesterol/phospholipid liposomes before assaying for cytotoxic function. Alterations affected cytotoxic capacity, albeit slightly differently in the different classes of killer cells.

The

Age (years)

compiled above clearly

emphasize the important role which can be attributed to lipids (and lipoproteins) in the course of an immune response; the discrepancies in the results and interpretations serve to make further research in this field even more challenging. It is beyond the scope of this review to discuss the in-vivo data supporting the concept of an effect oflipids on immune functions - suffice it to say that there have been two main approaches: (1) a study of the effect of different dietary lipids on normal immune function and/or disease; (2) a study of immune function in animals/patients with lipid/lipoprotein disorders resulting from disease or mc n~rmai agchlg pl ~ . A number of pitfalls accompany both approaches, among them the problem that lipids are precursors of steroid hormones and prostaglandins and the possibility of unconsidered dietary constituents (for example oxidized cholesterol) or deficiencies (for example, iron or zinc) so that observed effects on immunity may not necessarily be attributed to the lipids per se. However, the idea that this type of approach may be of clinical importance is supported by the data mentioned above concerning modulation of the tumour cell plasma membrane C/P1 to induce skin reactions to autologous tumour cells, and also by the results of a study on ageing humans where declining in-vitro Con A responsiveness of PBL correlated with decreasing membrane fluidity (Table III) and increasing serum C/P1. Acknowledgements

O u r own work cited above was supported by a grant from the Jubilfiumsfonds der Osterreicbischen Nationalbank (Jubilfiumsfondsprojekt No. 2016).

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Hemopoietic colony-stimulating factors Nicos A. Nicola and Mathew Vadas* Hemopoietic colony-stimulatingfactors (CSFs) areglycoproteingrowthfactors produced by many tissues in the body," they are essentialfor the survival, growth and differentiation of hemopoietic progenitor cells in vitro. In this article Nicos Nicola and Mathew Vadas discuss the classes of CSF that have now beenpurified (M-CSF, GM-CSF, G-CSF and Multi-CSF). Each is active in vitro at picomolar concentrations, but each can be distinguishedfrom the others by its unique molecularproperties and unique spectrum of biological activity. In addition to their proliferative effects, these regulators also appear able to stimulate functional activities in mature hemopoietic cells. Since the advent of semi-solid culture systems which allow the proliferation and differentiation of hemopoietic progenitor cells to occur in vitro1'2, it has become apparent that hemopoietic cell proliferation is under the absolute control of a family of regulators called colony-stimulating factors (CSFs). These regulators are active at extremely low concentrations (10-11-10-13 M) and are assayed by their ability to stimulate the development of colonies of differentiated cells from committed progenitor cells (colony-forming cells). It is now possible to grow hemopoietic colonies from nearly all the different types of committed progenitor cells 3. F u n c t i o n a l l y distinct members of the CSF family Since C SFs can be obtained from a multitude of sources including serum, urine, nearly all tissues and several cell lines, and since they exhibit multiple biological activities, it has not been simple to determine how many distinct members of the family exist. Moreover, determination of the molecular properties of a particular CSF has not Cancer Research Unit and *Experimental Allergy Laboratory of the Clinical Research Unit, Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Parkville, 3050, Victoria, Australia. © 1984,ElsevierSciencePublishersB.V.,Amsterdam 0167 4919/84/$02.00

always been helpful in determining the class to which it belongs since many CSFs apparently interact with other molecules in crude conditioned media and, as a consequence of their glycosylated nature, can exist in several forms of different size and charge. Nevertheless, it is now possible to clearly distinguish on both molecular and functional grounds four different classes of murine CSF (see Table I), each of which has been purified to homogeneity or near-homogeneity ~7. They can all stimulate granulocyte and/or macrophage colony formation in semi-solid cultures of murine bone marrow and are all remarkably stable glycoproteins requiring intact disulfide bonds for full activity. Each is active in vitro at low concentrations of 10-11-10-13 M. M - C S F has been purified from L cell-conditioned medium and is a 70 000 molecular weight glycoprotein consisting of two disulfide-bonded subunits 5. The subunits are biologically inactive and after removal of the carbohydrate chains have a molecular weight of 15 000 each 8. M - C S F stimulates predominantly 9or exclusively 10 macrophage colonies from murine bone marrow; this specificity has been confirmed by the binding of radiolabeled derivatives of M - C S F to various tissues. Antisera specific to M-C SF have been described which do not cross-react at all with the other three species of CSF ll.