Lipoprotein-induced inhibition of plaque-forming cell generation and natural killer cell frequency in aged donors

Ann. Immunol. (Inst. Pasteur) 1984, 135 C, 241-249

LIPOPROTEIN-INDUCED OF P L A Q U E - F O R M I N G AND

NATURAL

CELL

KILLER

IN A G E D

INHIBITION GENERATION

CELL

FREQUENCY

DONORS

by S. Antonaci (0, E. Jirillo (0, i . T. Ventura (0, A . R . G a r o f a l o (1) a n d L . B o n o m o

(8)

(1) Istitulo di Clinica Medica II, University o/Bari Medical School, Bari (Italy), (2) Istitulo di Microbiologia Medica, University o[ Bari Medical School, Bari (Italy), and (3) PatoIogia Medica III, University o[ Rome Medical School, Rome

SUMMARY The in vitro effects of human high-density, low-density and very-lowdensity lipoproteins on plaque-forming cell generation and natural killer cytotoxieity were evaluated in 40 aged donors. Lipoprotein pretreatment led to a significant inhibition in plaqueforming cell capacity displayed by either peripheral blood mononuclear cells or purified lymphocytes. As far as natural killer cytotoxieity is concerned, such pretreatment decreased the number of natural killer cells bound to targets, while the lytic efficiency was unaffected in both slCr-release and agarose-single-eell cytotoxie assays. These data suggest a role for lipoproteins in the impairment of cellmediated immunity in elderly individuals. KEY-WORDS: Plaque-forming cell, Natural killer cell, Lipoprotein, Cellmediated immunity, Ageing; Human, Impairment.

INTRODUCTION In recent years, the inhibitory effects of human lipoproteins (LP) on cellmediated immune functions have been extensively studied. In this regard, Manuscrit re~u le 19 janvier 1984, accept6 le 21 f6vrier 1984. Correspondence should be addressed to: Salvatore Antonaci, M. D., Clinica 1Vfedica II, Polielinico, 70124 Bari (Italy).

242

S. A N T O N A C I

AND

COLL.

high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) inhibit E-rosette formation as well as peripheral blood mononuclear cell (PBMC) mitogenesis [9]. As far as antibody response is concerned, VLDL injection in mice leads to a decrease in antibody synthesis [10]. Moreover, Curtiss et al. [11] have demonstrated a reduction of splenic and humoral haemagglutinin antibodies in mice inoculated with LDL. Additionally, recent data emphasize the in vitro influence of LP fractions on spontaneous plaque-forming cell (PFC) generation and the binding step of natural killer (NK) cytotoxicity in young healthy donors [3, 20]. By contrast, LP effects on cell-mediated immunity in aged individuals have been less evaluated, it is well known that aging is characterized by an impairment of both cell-mediated and humoral immunity [5, 17]. Since several factors have been suggested to modulate the immune response in aging [2, 12], it was of interest to evaluate the in vitro influence of different LP fractions on both PFC generation and N K cytotoxicity in elderly donors. Here we report t h a t in such individuals, LP treatment affects both spontaneous PFC capacity and NK cell frequency.

MATERIALS AND METHODS Donors. F o r t y aged v o l u n t e e r s (21 m a l e s a n d 19 females; age r a n g e 65-86 years, m e d i a n age 74), free of diseases affecting t h e i m m u n e s y s t e m or c a n c e r a n d t a k i n g no a n t i i n f l a m m a t o r y d r u g s or steroids, were considered in t h e s e studies.

PBMC isolation. P B M C were isolated f r o m h e p a r i n i z e d p e r i p h e r a l blood b y F i c o l l - H y p a q u e ( P h a r m a c i a , U p p s a l a , Sweden) d e n s i t y g r a d i e n t c e n t r i f u g a t i o n [6]. M o n o n u c l e a r cells w e r e r e s u s p e n d e d in R P M I - 1 6 4 0 (Eurobio, P a r i s , F r a n c e ) s u p p l e m e n t e d w i t h penicillin (100 I U / m l ) , s t r e p t o m y c i n (100 ~g/ml), 2 m M g l u t a m i n e a n d 5 % h e a t i n a c t i v a t e d l i p o p r o t e i n - d e p l e t e d foetal calf s e r u m ( L D F C S ; see below).

Peripheral blood lymphocyte (PBL) purification. A l i q u o t s of P B M C were r e s u s p e n d e d in H a n k s ' b a l a n c e d s a l t solution ( H B S S ) a t a c o n c e n t r a t i o n of 5 • 10~/ml, i n t r o d u c e d into p l a s t i c P e t r i dishes (Nunc, lqoskilde, D e n m a r k ) a n d i n c u b a t e d for 1 h a t 37 ~ C to allow m o n o c y t e adhesion [15]. A f t e r g e n t l e a g i t a t i o n , t h e n o n - a d h e r e n t P B L were r e c o v e r e d a n d r e s u s p e n d c d in R P M I 1640 s u p p l e m e n t e d w i t h penicillin (100 I U / m l ) , s t r e p t o m y c i n (100 ~g/ml), 2 m M

FCS HBSS HDL LDFCS LDL LP NK

= = = =

foetal calf serum. Hanks' balanced salt solution. high-density lipoprotein, lipoprotein-depteted foetal calf serum. = low-density lipoprotein. = lipoprotein. = natural killer.

PBL PBMC PBS 9PFC SRBC TBC VLDL

= = = = = = =

peripheral blood lymphocyte. peripheral blood mononuclear cell. phosphate-buffered saline. plaque-forming cell. sheep red blood cell. target-binding cell. very-low-density lipoprotein.

AGEING, LIPOPROTEINS, PFC AND NK FUNCTIONS

243

glutamine and 5 % LDFCS. In purified PBL, monocyte contamination was less than 3%, as assayed by the esterase technique [21]. Lipoprolein isolation. Fasting volunteers were selected for plasma collection. Pooled samples were used for LP isolation within 24h of collection. VLDL (d1.215) was prepared by layering KBr solution (d=1.215) over foetal calf serum (FCS) [4]. Following centrifugation at 260,000 g for 22 h, LP were discarded while LDFCS was dialysed as above described. Prelrealment o / P B M C or P B L with LP. Cell suspensions (4 • 106/ml) were incubated in RPMI-1640 supplemented with 5 % heat-inactivated LDFCS for 48 h at 37 ~C, 5 % CO2, in the absence or presence of 100 ~g/ml of HDL, LDL and VLDL, respectively. Thereafter, cells were extensively washed and used in the spontaneous PFC-system or cytotoxic assays. Cell viability, as determined by the Trypan blue exclusion dye test, was >95%. In another set of experiments, LP were directly added to uncultured cell suspensions before PFC assay. Spontaneous P F C generation. Protein A from Staphylococcus Cowan I (Pharmaeia, Uppsala, Sweden) was coupled to sheep red blood cells (SRBC), as previously described [2, 16]. Briefly, protein A-SRBC 25 ~1, 100 ~1 of the effeetor cell suspensions (2 • 106/ml), 25 ~1 of polyvalent (IgA-~IgG-rIgM) or monospecific antisera against human ~, y and chains (Behringwerke AG, Marburg, West Germany) from rabbit and 25 ~1 of freshly solubilized guinea-pig complement were added to 0.8 ml of agar (0.5~o Agar Noble, Difco Laboratories, Detroit, Michigan, USA) containing 0.05% DEAEdextran (Pharmacia, Uppsala, Sweden) and HBSS in a 46 ~ C waterbath. For plating, three individual 0.2-ml drops of the mixture were placed on three separate slides and each drop was immediately covered with 25 • mm glass coverslips, leaving an ultrathin gel. Plates were incubated for 5 h at 37 ~ C. PFC were then counted under indirect light and checked under a microscope for the presence of cells at the center of haemolytic spots. Thereafter, the number of plaques/10 G cells was calculated. N K cyloloxicily. K562, an established cell line (kindly provided by Dr Toru Abo, University of Alabama in Birmingham, Birmingham, Alabama, USA) was used as a target in the NK system. It was maintained as suspension cultures in RPMI-1640 supplemented with penicillin (100 IU/ml), streptomycin (100 9g/ml), 2 mM glutamine and 10% FCS (complete medium). 1) 5~Cr release assay. Cell suspensions (7 • 105) were washed in HBSS and labelled with 100 tzl of Na~51Cr04 (specific activity 0.5-1 mCi/ml; 3-20 ~gCr/ml; Radiochemical Center

244

S. ANTONACI AND COLL.

Amersham, England). After incubation for 1 h at 37 ~ C in humidified air containing 5% COs, the cells were washed and adjusted to a concentration of 50x103/ml. The assay was performed in duplicate, in conical plastic tubes, by mixing 5 x 103 labelled cells in 0.1 ml of complete medium with an equal volume of LP-treated or untreated PBL (effector/target cell ratios used were 60/1, 30/1, 15/1 and 5/1, respectively). A final volume (0.1 ml) of complete medium was added. The tubes were incubated for 4 h at 37 ~ C, 5% COs; afterwards, they were centrifuged for 10 min at 300 g and counted on a gamma counter. Cytotoxicity was expressed as % isotope released from target cells to medium; this value was corrected by subtracting the percentage of spontaneous isotope release in control tubes. The standard deviation of the difference in the duplicates was ~1.9. The spontaneous release from target cells ranged from 4 to 10%. 2) Agarose-single-cell cgtotoxicitg assay. NK activity of the same PBL suspensions was also evaluated by using the agarose-single-cell cytotoxicity assay originally described by Grimm and Bonavida [13]. Briefly, 100 ~l of effector cells and 100 ~l of target cells at 2 x 106/ml each were mixed, pelleted and incubated for l0 min at 37 ~ C and then resuspended by vigorous vortex treatment. To this conjugate mixture, 500 ~l of 0.5% agarose (Pharmacia) was added in RPMI-1640 which had been pre-cooled from 47 to 40 ~ C by 20-s exposure to room temperature. This cell-agarose mixture was plated onto Petri dishes and covered with complete medium. Control plates with target cells alone were also prepared. One set of plates was immediately harvested for background lysis, while duplicates were incubated at 37~ C for 4 h and harvested by aspirating off the medium, staining dead conjugates with Trypan blue for 5 rain, followed by two 5-min washes and fixation with 10/0 formaldehyde. Using an inverted microscope with a ><32 objective, the proportion of PBL bound to target cells was scored as the percentage of the total lymphocyte population by counting 300-400 PBL, and results were expressed as % target-binding cells (% TBC). Dead conjugated target cells were scored as percentage of the total number of conjugates by counting 50 to 75 conjugates, and the results were expressed as % dead conjugated target cells. The % NK cells was calculated as the product of % TBC times the percentage of dead conjugated target cells, since this represented the proportion of cells which could bind and kill NK-sensitive targets. Spontaneous target cell death, determined by Trypan blue exclusion, was found to be negligible.

Statistical analysis. Analysis was performed by means of the paired t test.

RESULTS L P p r e t r e a t m e n t of PBMC induced a significant impairment of PFC generation in aged donors. In fact, as shown in table I, H D L , L D L and V L D L inhibited spontaneous PFC capacity using either monospecific or polyvalent antisera. However, it has been previously demonstrated t h a t a d h e r e n t cells suppress spontaneous haemolytic plaque generation in elderly individuals, while m o n o c y t e depletion of PBMC suspensions leads to full recovery of spontaneous PFC capacity [2]. This is also confirmed by the present d a t a since, as illustrated in table II, purified P B L from the same aged donors exhibited a higher number of plaques when compared to u n f r a c t i o n a t e d PBMC. On the bases of these findings, it was of interest

AGEING, LIPOPROTE1NS, PFC AND NK FUNCTIONS

245

TABLE I. - - Effect of lipoproteins on PFC generation by PBMC from aged donors. Developing antiserum Anti- IgA

PBMC (control) 41•

(*)

HDL-treated PBMC

LDL-treated PBMC

YLDL-treated

22=t=5 (p<0.001) (**)

20=[=6 ( p < 0.001)

214-7 (p<0.001)

PBMC

Anti-IgG

44=t=10

214-8 (p<0.001)

224-9 (p<0.001)

304-11 (0.02>p>0.01)

Anti- IgM

524-12

304-11 (p<0.001)

34• 12 (p<0.001)

26s 15 (p<0.001)

Polyvalent ( I g A + IgG+ IgM)

574-10

464-9 (0.05>p>0.02)

404-11 (0.01>p>0.001)

364-10 (0.01>p>0.001)

(*) M e a n • of PFC/10 ~ cells. (**) Student's t test significance versus control.

TABLE II. - - PFC generation by lipoprotein-pretreated PBL from aged donors. Developing antiserum

PBL (control)

HDL-treated PBL

LDL-treated PBL

VLDL-treated PBL

Anti-IgA

864-15 (*)

71• (0.02>p>0.01) (**)

684-8 (0.02>p>0.01)

50j=10 (p<0.001)

Anti-IgG

96-t-ll

77=t=11 (0.01>p>0.001)

61=t=10 (p<0.001)

66~12 (p<0.001)

Anti-IgM

854-10

75• (0.05>p>0.02)

734-8 (0.05>p>0.02)

764-6 (0.05>p>0.02)

Polyvalent (IgA+ IgG+ IgM)

99 • 15

84 • 9 (0.05>p>0.02)

83 4- 6 (0.02>p>0.01)

81 =k6 ( 0 . 0 1 > p > 0.001)

(*) Mean• of PFC/10 6 cells. (**) Student's l test significance versus control.

to evaluate the L P effect on purified P B L in terms of spontaneous plaque formation. Table II shows that, despite adherent cell depletion, LP pret r e a t m e n t of P B L significantly affected PFC generation, although to a different degree according to the LP used. VLDL displayed the maximal inhibitory effect, while HDL and L D L suppressed haemolytie plaque production to a lesser extent. Furthermore, in control experiments, it has been observed t h a t neither the use of freshly isolated PBMC and/or P B L nor the addition of LP to cell suspensions immediately before PFC assay induced any significant difference in PFC frequency in comparison with LP-free cultured samples (data not illustrated).

246

S. ANTONACI AND COLL.

At the same time, the influence of LP on PBL-mediating N K activity was evaluated by using either a 51Cr-release assay or an agarose-single-cell cytotoxic system. As illustrated in table III, the different L P fractions did not affect spontaneous cytotoxicity in a UCr-release assay. The degree of cytotoxicity of LP-pretreated P B L was quite similar to that observed in untreated samples. Similar results were obtained using effector/target cell ratios ranging from 30/1 to 5/1 (data not shown). However, a significant decrease in NK-cell frequency was found following P B L exposure to LP. In fact, as shown in table IV, by using an agaroseTABLE Ill. - - Effect of lipoproteins on NK cytotoxicity from aged donors assayed by 51Cr-release assay.

Samples

% 51Cr release fronl K562

Ratio

Untreated P B L (control)

60/1

H D L - t r e a t e d PBL

60/1

36.7:k 13.3 (NS) (**)

PBL

60/1

38.2• (NS)

60/1

39.1• (NS)

LDL-trcated

V L D L - t r e a t e d PBL

49.0•

(*)

(*) Values are c o m p u t e d as m e a n + S D of net c y t o t o x i c i t y 40 donors. (**) Student's t test versus control (NS = not significant).

from

TABLE IV. - - NK activity assayed at the single-cell level following lipoprotein pretreatment of aged PBL.

TBC

Dead conjugated target cells

NK-cell frequency

Samples

(%)

(%)

(%)

U n t r e a t e d P B L (control)

10.2=k3.6 (*)

47.74-7.7 (**)

4.9~k2.1 (***)

H D L - t r e a t e d PBL

3.7•

42.2=59.1

1.5• (0.01>p>0.001) (****)

PBL

4.4•

41.6+5.9

1.8• (0.01>p>0.001)

3.9:k 1.5

40.9•

1.5i0.7 (0.01> p > 0.001)

LDL-treated

V L D L - t r e a t e d PBI,

(*) M e a n ~ S D of % target-binding cells. (**) M e a n • of % dead conjugated target cells. (***) M e a n • of % N K cell frequency. (****) Student's t test versus control.

AGEING, LIPOPFIOTEINS, PFC AND NK FUNCTIONS

247

single-cell cytotoxic system, TBC percentage was decreased in LP-treated fractions, while no significant differences were seen in t h e percentage of dead conjugated target cells. Therefore, by evaluating t h e frequency of NK cells, a low n u m b e r of these cells was found in the LP-pretreated samples when compared to the control.

DISCUSSION These data further confirm the inhibitory role of LP on cell-mediated immune functions, since LP pretreatment of effeetor cells reduces spontaneous PFC capacity in aged donors. In particular, this activity is usually depressed in elderly individuals, probably due to an exaggerated production of prostaglandins of the E series from adherent cells [2]. Such an effect is abrogated by either monoeyte depletion or indomethaein pretreatment of the whole mononuclear cell population [2]. LP pretreatment leads to an inhibition of spontaneous plaque formation regardless of t h e use of either PBMC or PBL in the system. This implies t h a t monoeytes are not involved in LP-indueed suppression, suggesting a direct effect of these substances on cells displaying PFC capacity. Conversely, NK cytotoxicity, assayed by a 51Cr-release assay, is unaffected by LP pretreatment. However, it is worthwhile mentioning that, when the NK phenomenon is evaluated at the single-cell level, L P dramatieally decrease the number of effeetor-to-target-bound cells, while the lytie capacity p er se is unmodified. Consequentially, the NK cell frequency is significantly reduced by such a procedure. One possible explanation for this discrepancy is the fact that, in the 51Cr-release assay, an effeetor cell ~ recycling ~ occurs, which may be responsible for a normal lyric capacity in spite of a low n u m b e r of active NK cells [19]. As far as the mechanism of LP-indueed inhibition is concerned, it has been previously observed that suppression of pre-replieative protein synthesis or sterol metabolism by VLDL inhibits mitogen-triggered DNA synthesis [7]. As an alternative hypothesis, it is conceivable that, since de novo cholesterol synthesis is required for some lymphocyte functions [7], the inhibition of cholesterol biosynthesis may be involved in the decrease in spontaneous PFC generation. This last mechanism may also account for the reduced NK-eell frequency through alterations in Na+/K + flux, changes in the binding of ligands to cell surface receptors and decreased cell-to-cell adhesiveness [8]. Moreover, these findings are in agreement with previous data on the LP-mediated inhibition of both PFC generation and NK-eell frequency in young donors [3, 20]. However, in contrast to young individuals, it should be emphasized t h a t the increased microviseosity of the cell membrane due to a high eholesterol/phospholipid molar ratio may account for reduced lymphocyte functions in the aging [181. Therefore, these data suggest t h a t the suppressive effect of L P might be an additional factor which contributes to the impairment of cell-mediated immunity in elderly individuals.

248

S. ANTONACI AND COLL.

R~SUM~ INHIBITION LIPOPROT]~INES-D I~PENDANTE DE LA PRODUCTION DE CELLULES FORMATRICES DE PLAGES ET DE LA FBI~QUENCE DES CELLULES N K CHEZ LE SUJET AG]~

Les effets in vitro des lipoprot6ines humaines H D L et L D L sur la production de cellules formatrices de plages et sur la cytotoxicit6 des cellules N K ont 6t6 6tudi6s chez 40 sujets ag6s. L'action des lipoprot~ines se t r a d u i t par une inhibition significative de la formation de plages, qu'il s'agisse de mononucl6aires sanguins p~riph6riques ou de pr6parations purifi~es de lymphocytes. L'action des lipoprot6ines sur les cellules N K se t r a d u i t par une r6duction du nombre des cellules N K fix~es sur leurs cibles tandis que leur activit6 lytique reste intacte, comme le montre l'essai au ~lCr aussi bien que le test de cytotoxicit6 en g61ose. Ces r6sultats sugg~rent que les lipoprot~ines j o u e n t un r61e dans l'alt~ration de l'immunit~ h m6diation cellulaire chez le sujet ag~. MOTS-CL~S : Lipoprot6ine, Cellule N K , Cellule formatrice de plage, Immunit6 cellulaire, A g e ; H o m m e , Alt6ration. ACKNOWLEDGEMENTS

This paper was supported in part by the (( Cousiglio Nazionale delle Ricerche ), (Progetto Finalizzato c(Medicina Preventiva e Riabilitativa ))-Sottoprogetto (cMeccanismi di Invecchiamento ~), Rome, Italy.

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249

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