Normal development of Lymphokine Activated Killing (LAK) in peripheral blood lymphocytes from hyperprolactinemic patients

Int. J. lmrnunopharrnac., Vol. 14, No. 7, pp. 1235- 1240, 1992. Printed in Great Britain.

0192-0561/92 $5.00 + .00 Pergamon Press Ltd. International Society for lmmunopharmacology.

NORMAL DEVELOPMENT OF LYMPHOKINE ACTIVATED KILLING (LAK) IN PERIPHERAL BLOOD LYMPHOCYTES FROM HYPERPROLACTINEMIC PATIENTS LINA MATERA,*+ ENRICA CICCARELLI,# GIAMPIERO MUCCIOLI,§ ALESSANDRA CESANO,* SILVIA GROTTOLI,t EMANUELAOBERHOLTZER* and FRANCO CAMANNI+ *Institute of Internal Medicine and * Department of Clinical Physiopathology, University of Turin, Corso Dogliotti 14, 10126 Turin; ~Institute of Pharmacology and Experimental Therapy, Corso Raffaello 30, 10125 Turin, Italy (Received lOApril 1992)

Abstract -- The effect of prolactin on the interleukin 2 (IL2)-driven development of Lymphokine Activated

Killing (LAK) by normal PBL and by PBL from hyperprolactinemic patients was investigated. Concentrations of PRL corresponding to the physiological serum levels of the hormone and to the Kd of the PRL receptors on NK cells (6-20 ng/ml, 0.3 - 1 nM) had no effect on the generation of LAK activity by normal PBL, whereas 100-200 ng/ml were slightly, although significantly, inhibitory. By contrast, PBL from 16 hyperprolactinemic patients developed levels of LAK activity comparable with those generated by PBL from age- and sex-matched normoprolactinemic donors.

Natural Killer (NK) cells have been assigned a defensive role in vivo on the basis of their ability to lyse a variety of virus or bacterial infected, as well as tumor, cells, without immunization and restriction to Major Histocompatibility Complex (MHC) products (Trinchieri, 1989). Upon activation with interleukin 2, (IL2) NK cells acquire the ability to kill previously resistant target cells (Grimm, Mazumder, Zhang & Rosenberg, 1982). Several reports agree that brain-linked hormones and neurotransmitters can affect NK activity. The observation that destruction of the tubero-infundibular dopaminergic (TIDA) system was associated with increased tumor growth in rodents (Bindoni, Belluardo, Licciarello, Marchese & Cirata, 1980) and the disappearance of NK function in mice (Cross, Markesbery, Brooks & Roszman, 1984; Forni, Bindoni, Santoni, Belluardo, Marchese & Giovarelli, 1983) suggested that uncontrolled prolactin (PRL) release could affect the NK cell system. However, further experimental and clinical observations have failed to unequivocally demonstrate a negative effect of hyperprolactinemia on NK cell function. We have observed recently that while high concentrations of PRL decrease the NK activity of normal peripheral blood lymphocytes (PBL) in vitro

(Matera, Cesano, Muccioli & Veglia, 1990; Matera, Bellone & Cesano, 1991), PBL from prolactinoma patients express normal NK function (Matera, Ciccarelli, Cesano, Veglia, Miola & Camanni, 1989a). Here we have extended our previous studies by exploring the effect of high concentrations of PRL on the IL2-driven acquisition of differentiated function by NK cells.

EXPERIMENTAL PROCEDURES

Patients. Thirty-three female patients (aged from 14 to 46 yr) with long lasting hyperprolactinemia (mean _+ S.D. PRL 76 _+ 24 ng/ml, range 2 7 - 2 1 4 ) and 26 sex- and age-matched normal subjects were studied. Plasma PRL levels were expressed as the mean of four samples during 24 h (at 8, 12, 16 and 20 h). No patient was on any drug known to affect either PRL levels or the immune system for at least 1 yr. CT scans revealed the presence of a prolactinoma in 14 patients, whereas in the remaining 19 patients the CT picture was normal. No patient with extrasellar invasion of the adenoma was included in this study.

*Author to whom correspondence should be addressed. 1235

1236

L. MATERAet al.

Reagents. Human PRL were provided by the Pituitary Hormone Distribution Program of the NIDDK, NIH (Baltimore, MD). ~25I-labeled human PRL were purchased from Dupont, NEN, Products Division, Italy. The specific activity of the labeled hormone varied from 33 to 48/xCi/gg. Hormonal determinations. Plasma PRL (normal values 3-20/xg/1) was evaluated by IRMA using reagents provided by SORIN Biomedica, Italy. The interassay and intraassay coefficients of variation were 3.1 and 6.9, respectively. Isolation o f PBL. Peripheral blood samples from patients and normal subjects were collected in tubes containing preservative-free heparin. Mononuclear cells were recovered from the interface of a Lymphoprep (Nyeegard, Norway) gradient. After three washings cells were suspended in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (Gibco, U.K.) (complete medium) and incubated in plastic flasks at 37°C for 2 h in a humidified incubator. Non-adherent cells (Peripheral Blood Lymphocytes, PBL) were collected and tested for generation of LAK activity and the '2q-PRL binding as described below. Generation o f L A K activity and cytotoxicity assay. PBL from 16 patients (Nos 1 - 1 6 ) were cultured at 106/ml in complete medium in the presence or absence of 1 0 0 U / m l recombinant human IL2 (Biogen, Behringwerke, F.R.G.). At the indicated culture times, cells were washed and tested for MHC-unrestricted cytotoxicity against the class I/class II MHC negative, K562 and HL60 cell lines (NK-susceptible and NK-resistant, respectively). Cytotoxic activity was measured using a standard 5~Cr release assay as described previously (Matera et al., 1989b). Briefly, 1 x 103 5~Cr-labeled target cells were added to the effector cells at the final effector:target (E:T) ratios of 100 : 1, 50 : 1, 25 : 1 and 12 : 1 in round-bottom microtiter plates (Linbro Scientific Co., Hamden, U.S.A.) in a final volume of 150 ~1. The plates were incubated at 37°C for 4 h and centrifuged at 1200 rev/min for 5 min. Aliquots (100 tA) of supernatant were collected and counted in a gamma counter. All experiments were performed in triplicate and the percentage of 5tCr release was expressed as: ( E - S ) / ( M - S ) x 100, where E is counts/rain release in the presence of effector cells; S is counts/min spontaneously released by target cells incubated with medium alone and M is counts/min contained in 100 #1 resuspended target cells. The cytotoxic activity was determined by plotting the percentage of 51Cr release against the E : T ratio and expressed as Lytic Unit (LU) per 108 PBL (Pross, Baines, Rubin, Shragge & Patterson, 1981), so as to

transform the 5~Cr release dose response data to linearity. A LU is defined as the number of effector cells required to produce 30070 specific cytotoxicity of 1 × 103 target cells. PRL binding assay. The PRL binding assay was performed on the blood of 17 patients (patients Nos 1 7 - 33) as described previously (Bellussi, Muccioli, Ghe' & Di Carlo, 1987). Briefly, 2 × 10~' PBL were incubated in triplicate with approximately 60,000 counts/rain ( 0 . 6 - 0 . 8 ng) of tzsI-PRL in a final volume of 0.5 ml Dulbecco Minimal Essential Medium (DMEM) containing 0.1070 BSA. The incubation was carried out at 37°C for 6 h, a condition shown to favor optimal binding of t251-PRL to human lymphocytes (Russell, Kibler, Matrisian, Larson, Poulos & Magun, 1984). Cells were washed twice in DMEM plus BSA and the tubes containing the cell pellet, together with triplicate blank tubes, counted. The radioactivity found in the blank tubes (0.2-0.3070) was subtracted from the binding values. Unless otherwise indicated, specific binding was calculated as the difference between binding in the absence and in the presence of excess unlabeled PRL (2~g/ml) and expressed as a percentage of the total counts added to each tube for 2 × 106 cells. Scatchard analysis of PRL binding to PBL was also performed by transformation of binding data obtained by competition studies with increasing concentrations of unlabeled PRL mixed with a fixed amount of tracer. The dissociation constant (Kd) and the binding capacity were determined for PRL concentrations between 1.6 and 60 ng/ml. Total PRL receptors were evaluated as described previously (Muccioli, Guardabassi, Pattono & Genazzani, 1988) by exposing lymphocytes to 4 M MgC12. This treatment results in a complete dissociation of specifically bound '25IhPRL to lymphocytes without affecting subsequent rebinding ability. Lymphocytes were subdivided into two fractions and treated as follows: fraction I was resuspended in 4 M MgC12 (0.2 ml/2 x 106 cells); fraction II was resuspended in binding medium and served as a control (free PRL receptors). After 5 rain at 20°C, cells were washed twice with cold DMEM and binding of ~25I-hPRL was assayed as described above. Statistical analysis. The LU values (logarithmically transformed to reach near normal distribution) were compared by paired (effect of PRL treatment on LAK activity) or unpaired (LAK activity of patients vs LAK activity of normals) Student's test. ~25I-PRL binding was also compared by paired (effect of PRL on ~25I-PRL binding) or unpaired (t25I-PRL binding of patients vs normal PBL) Student's test.

1237

Normal Development of LAK 1200

A

1600 ~0

1000

~

6oo •~ o

1400 ~

1200

~"

1000

I !

B % m

600 ~ 0 ~ v ~

4ooi

~

200

I"i,i 7

0

o

600 |

. ',

Patients

N0rmals

600 400 200 0

0 0

0 100

6 100

12 100

25 100

50 100

100 100

200 100

16oo ~0

B

..]

1400 •

=21,oo. ~

i~o 0

PRL (ng/ml) IL2 {U/I~)

B 1400 ' 0

lOOO.

1200 '

i %l

600.

-J >-

1000 •

600,

--

coo.

400 -

I.0

600 .

v,

400 '

PUl Z

200

200 ,

--.|.-I

:•

1

Patients

N or ma l s

00 0

100

6 100

12 100

25 100

50 100

100 200 100 100

PRL (ng/ml) IL2 (U/ml)

Fig. I. Inhibitory effect of high concentrations of PRL on development of LAK activity by IL2-stimulated normal PBL. PBL from normal donors were pre-incubated for 16 h with or without PRL, before culture with or without IL2 (100 U/ml). After 4 days cells were tested for cytotoxicity towards the NK-susceptible K562 (A) and the NKresistant HL60 (B) cells. Results refer to five separate experiments using PBL from different normal donors and are expressed as mean _+S.D. LU/108. One LU is the number of effectors required to induce 30% lysis of 103 target cells. RESULTS

Effect of PRL on the development of L A K activity from normal PBL. A f t e r a 4-day i n c u b a t i o n with IL2 (100 U / m l ) n o r m a l P B L killed the NK-sensitive cell line K562 with a m u c h higher efficiency t h a n did control PBL cultured in IL2-free medium [Fig. I(A)]. In a d d i t i o n , IL2-treated P B L were very effective at killing the previously resistant H L 6 0 cell line [Fig. I(B)]. A 16-h p r e - i n c u b a t i o n with P R L h a d a negative effect o n the s u b s e q u e n t d e v e l o p m e n t o f L A K activity against b o t h targets w h e n the h o r m o n e was used at c o n c e n t r a t i o n s o f 100 (10.3% i n h i b i t i o n for K562, P < 0 . 0 5 ; 8.5% i n h i b i t i o n for HL60, P < 0 . 0 5 ) a n d 200 n g / m l (31% i n h i b i t i o n for K562, 1°<0.005; 2 2 . 9 % i n h i b i t i o n for HL60, P<0.02). I n h i b i t i o n was n o t due to aspecific d e a t h o f effector cells, as s h o w n by the u n c h a n g e d survival o f P R L -

0

Fig. 2. Generation of LAK activity by PBL from hyperprolactinemic patients and normal subjects. Peripheral blood lymphocytes (PBL) from 16 patients (Nos 1 - 16) and 16 age- and sex-matched normal donors were cultured for 4 days with IL2 (100 U/ml) and used as effectors of cytotoxicity against the K562 (A) and HL60 (B) cell lines. Results are expressed as net LUs (LUs of stimulated minus LUs of unstimulated cells). K562 and HL60 cytotoxicity in IL2-unstimulated cultures were 32 + 12 and 3 __+1, respectively, in patients and 28 + 12 and 2 + 0, respectively, in normal donors. Serum PRL levels were 68 _+ 45 range 27 - 182 ng/ml for patients and 7 _+ 3 range 2 - 13 ng/ml for normal donors.

treated PBL. In addition, the 5~Cr u p t a k e a n d s p o n t a n e o u s release of K562 a n d H L 6 0 cells was not affected after a 4-day culture in the presence o f 200 n g / m l P R L (not shown).

L A K response of PBL from hyperprolactinemic patients and from normal subjects. C o m p a r a t i v e analysis of the m e a n net L U values o f the day-4 L A K cultures showed n o difference between hyperprolactinemic patients a n d n o r m a l individuals (1220_+ 312 vs 1182_+ 321, P > 0 . 5 for K562; 835 -+ 220 vs 880 + 231, P > 0 . 5 for HL60) [Figs 2(A) a n d (B)]. The regression analysis showed n o correlation between the s e r u m P R L level in patients a n d their capacity to develop L A K activity as

1238

L. MATERAet al.

Table 1. Effect of PRL in vitro on free and total PRL receptors of PBL from normal donors PRL binding parameters Free receptors

Total receptors

h-PRL added (ng/ml)

Bmax*

Kd*

Bmax

Kd

Experiment I 0 20

472 +_ 57t 426 _+ 61

2.7 + 0.35 2.6 _+ 0.30

483 _ 44 435 _+_46

2.5 _+ 0.23 2.7 _ 0.17

Experiment II 0 100

544 _+ 34 270 _+ 49*

2.8 _+ 0.17 2.7 + 0.11

563 _+ 41 271 _+ 36*

3.0 _+ 0.28 2.6 _+ 0.17

Cells were pre-incubated for 3 h with or without hPRL, as indicated, before being used for PRL-binding studies. *Brnax (sites/cell) and Kd (× 10 ,0 M) were obtained by Scatchard analysis. *Values are mean + S.D. of three determinations for each experiment. *P<0.05 compared with control. Table 2. PRL serum levels and PRL receptor binding characteristics of PBL from normal and hyperprolactinemic patients

Patients Normal n =6 ~ Hyperprolactinemic n= 6

PRL serum levels (ng/ml)

t25Ih-PRL binding parameters B*

Bmax t

Kd t

6 _+ 2.1'

0.91 _ 0.06

743 _+ 66

2.3 +_ 0.14

83 + 33~

0.82 + 0,08

617 + 61

2.1 _ 0.73

*Specific binding values (B) is expressed as percent of total counts/min added per 2 × 106 cells. *Bmax (sites/cells) and Kd (× 10-~0 M) were obtained by Scatchard analysis. *Values are mean _ S.D. ~n = number of patients. ~P<0.05 vs normal subject. evaluated by the net L U values (r = 15). Both in patients and in n o r m a l d o n o r s maximal expression o f L A K activity was observed after 4 days in culture with IL2 (not shown).

Effect o f normal and high concentrations o f P R L on modulation o f the P R L receptors. We then considered the possibility that the refractoriness o f L A K precursors f r o m hyperprolactinemic patients to the depressive effect o f a b n o r m a l l y high circulating levels o f the h o r m o n e could be a consequence o f the reduced expression o f specific P R L binding sites. Indeed, a 3-h exposure to high (100 n g / m l ) , but not to physiological concentrations o f P R L , was s h o w n in preliminary experiments (Table 1) to p r o d u c e a 50°7o decrease o f b o t h total and free P R L receptors on lymphocytes f r o m n o r m a l d o n o r s . H o w e v e r , the n u m b e r o f these receptors was not significantly

decreased in P B L f r o m 6 hyperprolactinemic patients as c o m p a r e d with sex- and age-matched normoprolactinemic individuals tested simultaneously (Table 2). F u r t h e r m o r e , the specific binding o f 125I-hPRL to P B L f r o m a n o t h e r 11 h y p e r p r o lactinemic patients was not different f r o m that o f P B L f r o m 4 simultaneously tested n o r m a l d o n o r s (0.78 ± 0.2 and 0.92 ± 0.1, respectively, P>0.05) (not shown). N o correlation was f o u n d in the binding parameters and P R L serum levels in all the patients examined (r -- 12). DISCUSSION

W e show here that in vitro exposure to high c o n c e n t r a t i o n s o f P R L significantly decreases the ability o f n o r m a l N K cells to develop IL2-induced

Normal Development of LAK LAK activity. Generation of LAK activity is secondary to interaction of IL2 with the IL2-receptor (R) beta chain (p70), constitutively expressed by resting NK cells (Siegel, Sharon, Smith & Leonard, 1987; Phillips, Takeshita, Sugamura & Lanier, 1989). Thus, one reason for the suppressing effect of PRL on the generation of LAK activity could be its down-modulating effect on the expression of the IL2-binding molecule. However, concentrations of PRL as high as 200 ng/ml did not modify the frequency of cells expressing the p70 IL2R subunit (data not shown). Thus, pre-incubation with PRL seems to affect some intracellular events distal to the IL2/IL2-R p70 binding and directly involved in the activation of the lytic machinery, such as increased synthesis of perforins. Whichever the mechanism underlying the PRLinduced decrease of the IL2-activated cytotoxicity, it does not seem to be effective during prolonged exposure in vivo of NK cells to abnormally high levels of the hormone. This is suggested by the observation of a normal response to IL2 by PBL freshly isolated from hyperprolactinemic patients. One could argue that the average levels of PRL in the blood of patients were lower than those affecting the LAK response in vitro (100 ng/ml). However, no correlation was found between PRL levels and the LAK response in the range of serum PRL concentrations ( 2 7 - 1 8 2 ng/ml) exhibited by the patients. Opposite consequences of hyperprolactinemia on the in vitro and in vivo human NK cell function have already been described by us and by other groups. A functional and numerical impairment of the NK system has been reported by Gerli et al. (Gerli, Rambotti, Nicoletti, Orlandi, Migliorati & Riccardi,

1239

1986) in hyperprolactinemic patients, although these authors failed to confirm the inhibitory effect of PRL in vitro. In contrast, we have demonstrated a strong decrease of NK activity in normal PBL incubated with high (100-200 ng/ml) concentrations of PRL (Matera et aL, 1990, 1991) and normal NK activity in PBL from hyperprolactinemic patients (Matera et al., 1989a). Besides the increased PRL release, altered levels of other neuropeptides and hormones may occur in prolactinoma patients with hypothalamic damage. The fact that such patients were excluded from our studies through a careful radiological screening, may perhaps explain the discrepancy between our and others' data. The existence of different forms of PRL (Haro, Lee, Singh, Bee, Markoff & Lewis, 1990) may also create a discrepancy in the definition of hyperprolactinemia in different laboratories. The apparent refractoriness of LAK progenitors from patients to the inhibitory effect of high concentrations of PRL, observed here, was not due to down-regulation of PRL receptors, as shown by the results of ~2~I-PRL binding studies. In conclusion, our data demonstrate that chronic exposure to high levels of circulating PRL does not modify the IL2-responsive state of NK cells. Further studies on other situations of in vivo hyperprolactinemia are needed to establish whether the PRL-mediated inhibitory effects observed in vitro have any in vivo counterpart. Acknowledgements - - This work was supported by Ricerca

Finalizzata Piemonte n. 150. Silvia Grottoli and Emanuela Oberholtzer are recipients of fellowships by Regione Piemonte and Fondazione Bossolasco, respectively.

REFERENCES

BELLUSSI, G., MUCCIOLI,G., GHE', C. & DI CARLO, R. (1987). Prolactin binding sites in human erythrocytes and lymphocytes. Life Sci., 41, 951- 959. BINDONI,M., BELLUARDO,N., LICCIARELLO,S., MARCHESE,A. E. • CIRATA,F. (1980). Growth of Yoshida ascite tumor in rat after radiofrequency destruction of the tuberoinfundibolar region of the hypothalamus. Neuroendocrinology, 30, 88 - 94. CROSS, R. J., MARKESBERY,W. R., BROOKS,W. H. & ROSZMAN,T. L. (1984). Hypothalamic immune interactions: neuromodulation of natural killer activity by lesioning of the anterior hypothalamus. Immunology, 51, 399- 416. FORNI, G., BINDONI,M., SANTONI,A., BELLUAROO,N., MARCHESE,A. E. & GIOVARELLI,M. (1983). Radiofrequency destruction of the tuberoinfundibular region of the hypothalamus permanently abrogates NK cell activity in mice. Nature, 306, 181 - 186. GERL1,R., RAMBOTTI,P., NICOLETTI,I., ORLANDI,S., MIGLIORATI,G. & RICCARDI,C. (1986). Reduced number of natural killer cells in patients with pathological hyperprolactinemia. Clin. exp. Immun., 64, 399- 406. GRIMM,E. A., MAZUMDER,A., ZHANG,H. Z. & ROSENBERG,S. A. (1982). Lymphokine activated killer cell phenomenon. J. exp. Med., 155, 1823- 1841.

1240

L. MATERA et al.

HARO, L. S., LEE, O. W., S1NGH, R. N. P., BEE, G., MARKOFF, E. &: LEWIS, U. J. (1990). Glycosylated human prolactin: alteration in glycosylation pattern modify affinity for lactogen receptor and values in prolactin radioimmunoassay. J. clin. Endocr. Metab., 71, 379-383. MATERA, L., BELLONE, G. & CESANO, A. (1991). Prolactin and the neuroimmune network. Adv. Neuroimmun., 1, 158 - 172. MATERA,L., CESANO,A., MUCCIOLI,G. t~ VEGL|A, F. (1990). Modulatory effect of prolactin on the DNA synthesis rate and NK activity of large granular lymphocytes. Int. J. Neurosci., 51, 265-267. MATERA, L., CICCARELLI, E., CESANO, A., VEGLIA, F., M1OLA, C. ~: CAMANNI, F. (1989a). Natural killer activity in hyperprolactinemic patients. Immunopharmacology, 18, 143 - 146. MATERA, L., FOA, R., MASSAIA, M., GIOVARELEI, M., VEGLIA, F., CESANO, A., LUSSO, P., PIAZZA,A. & SANTOLI,D. (1989b). B cells from chronic lymphocytic leukemia (CLL) patients are strong inducers of proliferation and major histocompatibility complex (MHC)- unrestricted [natural killer (NK)-like] cytotoxicity in normal T-lymphocytes. J. clin. Immun., 9, 329-337. MUCCIOLI, G., GUARDABASSI,A., PATTONO, P. • GENAZZANI,E. (1988). Further study on the changes in the concentration of prolactin-binding sites in different organs of Xenopus laevis male and female, kept under dry conditions and then returned to water (their natural habitat). Gen. Compar. Endocr., 74, 4 1 1 - 417. PHILLIPS, J. H., TAKESHITA, T., SUGAMURA, I(. ~¢ LANIER, L. L. (1989). Activation of natural killer cells via the p75 interleukin 2 receptor. J. exp. Med., 170, 2 9 1 - 296. PROSS, H. F., BAINES, M. G., RUB1N, P., SHRAGGE,P. & PATTERSON, M. S. (1981). Spontaneous human lymphocytemediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity. J. clin. Immun., 1, 5 1 - 6 4 . RUSSELL, D. H., KIBLER, R., MATRISIAN, L., LARSON, D. F., POULOS, B. & MAGUN, B. E. (1985). Prolactin receptors on human T and B lymphocytes: antagonism of prolactin binding by cyclosporin. J. Immun., 134, 3027-3031. SIEGEL, J. P., SHARON, M., SMITH, P. L. & LEONARD, W. J. (1987). IL-2 receptor beta chain (p70): role in mediating signals for LAK and proliferative activities. Science, 238, 7 5 - 78. TRINCHIERI, G. (1989). Biology of natural killer cell. Adv. lmmun., 47, 187-376.