In vivo enhancement of NK-cell activity by thymopentin

Int. Z lmmunopharmac., Vol. 12, No. 2, pp. 1 9 3 - 1 9 7 , 1990. Printed in Great Britain.

0 1 9 2 - 0 5 6 1 / 9 0 $3.00 + .00 International Society for lmmunopharmacology.

I N VIVO E N H A N C E M E N T OF NK-CELL ACTIVITY BY THYMOPENTIN C. HU,* L. RADELLI, R. SCORZA, P. BONARA, R. PEREGOt and G. FANTUZZI Institute of Internal Medicine, Infectious Diseases and Immunopathology, University of Milan and tMedical Department, Italfarmaco S.P.A., Milan, Italy (Received 4 January 1989 and in final form 10 September 1989)

- - Natural killer (NK) activities of fresh and IL-2-precultured peripheral blood mononuclear cells (PBMC) were investigated in 13 elderly subjects before and after thymopentin (TP-5) treatment and in 12 age-matched controls. The NK activity of fresh PBMC was found to be significantly higher in subjects given TP-5 at 50 mg s.c. three times per week for one month. Conversely, this type of treatment with TP-5 did not significantly modifie the cytotoxic capacity of IL-2-precultured PBMC.

Abstract

Natural killer (NK) activity against virus-infected and tumor cells is displayed by a distinct group of peripheral blood mononuclear cells (PBMC) with morphological characteristics of large granular lymphocytes (LGL) (Herberman & Ortaldo, 1981; Timonen, Ortaldo & Herberman, 1981). There is also considerable evidence that LGL may play a critical role in the in vivo resistance against bone marrow allografts and in the control of tumor growth and metastatization (Kiessling, Hochmann, Haller, Shearer, Wigzell & Cudkowicz, 1977; Herberman, 1983; Warner & Dennert, 1982). Activity of LGL can be modulated by a variety of agents (Herberman, Brunda, Cannon, Djeu, NunnHargove, Jett, Ortaldo, Reynolds, Riccardi & Santoni, 1981; Ortaldo, Mason, Rehberg, Kelder, Harvey, Osheroff, Pestka & Herberman, 1983), among which is interleukin 2 (IL-2) that was recently shown to support the continuous growth of LGL and to boost their NK activity whether employed alone (Domzig, Stadler & Herberman, 1983) or in association with IFN (Dempsey, Dinarello, Mier, Rosenwasser, Allegretta, Brown & Parkinson, 1982). These lymphokine-activated cells (LAK-cells) are potent cytotoxic cells that seem to be important in in vivo immunotherapy of solid tumors (Rosenberg, Lotze, Muul, Chang, Avis, Leitman, Linehan, Robertson, Lee, Rubin, Seipp, Simpson & White, 1987).

It has been proposed that the enhancement of T-cell function in immunocompromised hosts induced by thymic hormones is at least in part mediated by an increase in IL-2 production (Meroni, Barcellini, Frasca, Sguotti, Borghi, De Bartolo, Doria & Zanussi, 1987). This led us to investigate the in vivo effects of treatment with thymopentin (TP-5), a pentapeptide endowed with thymic hormone-like activity (Goldstein, Scheid, Boyse, Schlesinger & Van Wauwe, 1979) on NK and LAK activities in healthy elderly volunteers. Results presented here demonstrate that in vivo TP-5 treatment is associated with a significant increase in the NK activity of fresh PBMC but not in that of IL-2-pre-exposed PBMC. EXPERIMENTAL

PROCEDURES

Subjects

Subjects were 25 healthy institutionalized aged volunteers (21 women and 4 men, aged 65 - 94 years; mean age +__S.D. 79 _+ 8.2). They were randomly divided into two groups, and 13 subjects were treated with thymopentin (TP-5) for one month (50 mg s.c. three times per week), whereas 12 were left untreated. The subjects gave informed consent. At the time of the study none of the subjects had neoplastic, infectious or autoimmune disease, nor were they under medication with recognized activity on the immune system.

*Author to whom correspondence should be addressed at: Clinica Medica I, pad. Granelli Ospedale Policlinico via F. Sforza, 35 20122 Milano, Italy. 193

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C. Hu et al.

Peripheral blood fractionation

mononuclear

cell

(PBMC)

PBMC were separated from heparinized fresh blood by centrifugation on F i c o l l - H y p a q u e , using the method of Boyum (1968) and resuspended in RPMI-1640 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS, Gibco, Grand Island, NY), 2 mM glutamine (Eurobio, Paris, France) and antibiotics (100 I U / m l penicillin and 100/~g/ml streptomycin) (Eurobio, Paris, France).

N K cell activity A 18 h 51Cr release test was employed with K562 cells as targets. In our experience, specific cytotoxicity results using K562 targets are similar for both 4 and 18 h NK assays, as also previously described by Pross, Baines, Rubin, Shragge & Patterson (1981). Briefly, 5~Cr-labelled target cells were incubated with various concentrations of effector cells in a total volume of 200 ~1. Effector to target (E:T) ratios used were 50:1, 25:1, 12.5:1, and 6.25:1, and all tests were carried out in triplicate. After 18 h incubation, the supernatant was collected and counted for radioactivity. Specific NK cytotoxicity was calculated as follows:

counts/min in supernatant%NK = counts/min spontaneous release × 100 counts/min total - counts/min spontaneous release

Data are expressed either as mean specific cytotoxicity values at different E:T ratios or in terms of mean lytic units (L.U.); one L.U. is defined as the number of effector cells needed to produce 80% specific cytotoxicity above the baseline value.

Statistical analysis Comparisons between data before and after TP-5 treatment, or before and after IL-2 preincubation were made by the Student's t-test for paired data. The calculation of the correlation coefficient, r, was made by linear regression analysis.

RESULTS

Effects o f TP5 treatment on the N K activity of fresh PBMC No significant variations were observed when the NK activity of age-matched untreated subjects was assessed at the time zero (To) and after 1 month (T0. In fact in all subjects the L.U. values did not vary more than 10%. As shown in Fig. 1A the NK activity of fresh PBMC in TP5-treated subjects was significantly increased after treatment. In fact, before treatment the mean (_+ S.E.) cytotoxicity value were 274.6 .+ 24.1 L.U./108 cells, whereas after 1 month of drug treatment the mean value reached 343 _+ 25.7 (P<0.05). The individual cytotoxicity values measured to the 50:1 E:T ratio are presented in Fig. 1B; it can be seen that an increase of more than 20% above baseline was observed in eight out of thirteen subjects. The TP5-induced increase in NK activity was highest in those subjects with the lowest basal lytic values. In fact, as shown in Fig. 2, a significant negative correlation was found between basal NK activity expressed as L.U./108 cells (Fig. 2A) or as per cent 5'Cr release at 50:1 E:T ratio (Fig. 2B), and the per cent variation of this activity (delta %) after treatment (r = - 0 . 9 1 , P<0.001, r = - 0 . 7 9 , P<0.001, respectively).

Effect o f TP-5 treatment on the N K activity o f IL-2 preincubated PBMC Pretreatment o f P B M C with IL-2 Pretreatment of PBMC was performed according to Seki, Ueno, Taga, Matsuda, Miyawaki & Taniguki (1985). Briefly, PBMC (1 × 106/ml) were cultured for 48 h in a humidified atmosphere of 5°7o CO2 in air at 37°C; for LAK induction, human recombinant IL-2 (Biogen, Switzerland) was added at 100 U / m l in flat-bottomed 24-well tissue culture plates (Flow Laboratories, The Netherlands). After incubation, the cells were washed twice, their viability assessed by the Trypan blue dye exclusion test and tested for cytotoxic activity against K562 cells as described above.

In controls as well as in subjects belonging to the experimental group, in vitro preincubation with IL-2 significantly enhanced the spontaneous cytotoxic activity of PBMC (Fig. 3A). Mean basal cytotoxic values were in fact 274.6 + 24.1 L.U ./ 1 0 ~ cells, whereas after IL-2 preincubation the mean calculated value was 335.1 + 27.5 (P<0.05). Again, a significant negative correlation was found between the basal NK activity and the per cent variation of this activity after incubation with IL-2 (r = - 0 . 8 7 ; P<0.001) (Fig. 3B), indicating that IL-2-induced increases of NK activity were highest in those subjects with lowest basal lytic values. The increase

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Fig. 3. A. NK activity (expressed as mean lytic units/1 08cells, L.U./1 08 cells) of the experimental group before (b) and after (IL-2) in vitro preincubation with IL-2. B. Correlation between basal NK activity and per cent variations (delta% b-IL-2) after in vitro preincubation with Ik-2, in the experimental group before treatment.

in NK activity induced by in vitro preincubation with IL-2 was quantitatively similar to that seen after in vivo treatment with TP-5. In fact, a significant positive correlation was found between the per cent variation of lyric activity with in vivo TP-5 and in vitro IL-2 (r = 0.88; P<0.001). At variance with what was seen for NK activity of flesh PBMC, the TP-5 treatment employed was not associated with significant increases viz-gt-viz controls in the cytotoxicity values of IL-2-preincubated PBMC. DISCUSSION The results presented herein indicate that the administration of TP-5 to healthy elderly individuals is associated with a significant enhancement in NK cell activity, thus extending the results of previous studies showing that in vitro TP-5 increases NK activity of mouse bone marrow NK cells (Fiorilli, Sirianni, Sorrentino, Testi & Aiuti, 1983). It is accepted that NK cell activity is highly variable among individuals (Pross & Baines, 1982) and that genetic factors contribute to such a variability (Dubey, Yunis, Leslie, Mehta & Yunis, 1987). The negative correlation observed in this

study between basal NK activity and the extent of its variation after TP-5 treatment indicates that this drug acts mainly in subjects with NK activity below their maximal level. Indeed, in vivo TP-5 was able to restore NK activity in one patient with severe immunodeficiency (Fiorilli, Sirianni, Pandolfi, Quinti, Tosti, Aiuti & Goldstein, 1981). Recently, similar observations were reported by Chisesi, Capnist, Rancan, Pellizzari & Vespignani (1988) who described an increase in the value of the absolute lymphocyte numbers after treatment with thymic substances in Hodgkin's lymphoma patients with severe lymphopenia, but not in those with mild or no lymphopenia. The mechanism(s) of the immunopotentiating activity of TP-5 are still a matter of discussion. Recent data indicate that the in vivo administration of TP-5 to immunocompromized aged subjects enhances the production of IL-2 (Meroni et al., 1987), a factor which is well known for its capacity to modulate NK cell activity (Domzig et al., 1983). Results here described appear to give indirect support to the contention that IL-2 is indeed involved in the enhancement of NK cell activity observed after TP-5 treatment. In fact, the levels of NK activity of PBMC measured after TP-5 treatment

Effects of Thymopentin on NK Cell Activity did n o t differ f r o m those o b s e r v e d for the same subjects b e f o r e t r e a t m e n t w h e n their cells were tested for cytotoxicity after p r e i n c u b a t i o n with IL-2. In line with such a possibility, a significant positive

197

c o r r e l a t i o n was f o u n d in this study between the per cent variations o f lytic activity after TP-5 t r e a t m e n t a n d after IL-2 in vitro p r e i n c u b a t i o n , o b t a i n e d in subjects before t r e a t m e n t .

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