Natural killer activity against human K562 tumour cells during Plasmodium cynomolgi malarial infection of rhesus monkeys

FEMS Mio'obiologyImmunology64 0990) 121-124 Published by Elsevier

121

FEMS1M 00110

Natural killer activity against human K562 tumour cells during Plasmodium cynomolgi malarial infection of rhesus monkeys Q.B. S a x e n a a n d S. Biswas Malaria RexearchCentre.Delhi. India Received20 December1989 Revisionreceived29 March 1990 Accepted9 April 1990 Key words: Natural killer cell; Malaria; Plasmodium spp.

I. S U M M A R Y This study assessed the natural killer (NK) cell activity profile during Plasmodium cynomolgi infection in rhesus monkeys. There was a significant decrease in the N K cell activity in the peripheral blood leukocytes of iMected monkeys during the early, ascending phase of infection. However, as the parasite load d e c r e ~ d , N K 'cell activity returned to normal levels. "ilxis could be correlated with the peak increase in b mphocyte counts. Tl.ls indicated that a decrease in N K cell activity observed at an earlier stage during an active P. vivax malarial infection was a tempor,u'y phenomenon.

2. I N T R O D U C T I O N Natural killer (NK) cells constitute a naturally occurring defence mechanism which m a t play an important role in efiminating spontaneously arising tumour cells [1]. There is some evidence which indicates a possible role for N K cells in immumty

Correspondence to: Dr. Queen B. Saxena. Present address: Pittsburgh Cancer Institute. 200 Meyran Avenue, Pittsburgh. PA 15213,U.S.A.

to parasites. Solomon et aL [2] found evidence for a possible protective role of N K cells in rat malaria. Eugui and Allison [3] reported a correlation between N K cell numbers and susceptibility to the malarial parasite in different strains of mice, however, a direct role for N K cells in malarial immunity has not been established. Indeed, Wood and Clark [4] have suggested that N K cells may be unimportant in malarial immunity. One report [5] on the status of the changes in h u m a n malaria does not give a clear picture of the changes in N K cell numbers during the course of the disease. Previously, it was shown that N K cell numbers in peripheral blood decreased during the active phase of h u m a n P. vivax infection [6]. In the present study, the monkey malaria model was used to gain an understanding of the changes in the N K cell population during different phases, i.e. ascending (AP), descending (DP), and recovery (RP), of the infection. 3. MATERIALS A N D M E T H O D S 3.1. Experimental infection of rhesus monkeys with P. cynomolgi Healthy adult rhesus (Macaca mulatta) were purchased locally and quarantined for two weeks

0920-8534/90/.$03.50 © 1990 Federation of European Microbiological:~ocieties

t22 before initiating a P. cynomolgi (from National Institute of Communicable Diseases, Delhi) infection by the transfer of 1 × I0 e ittfected erythrocytes from an infected monkey to a healthy monkey. 3.2. Parasitaemia ¢md leukocyte monitoring Parasitaemla and the differential count of leukocytes were monitored at different time perlods following infection up to 28 days, Parasitaemia was determined from JSB (Iaswant Singh Bhattachatya strain) stained blood smears as described [7]. The number of parasitized erythrocytes in 1 × 103 RBCs was counted for the calculation of percent parasitacmia. A differential leukocyte count was also made from the same blood smear by counting the different cell populations in at least 200 fields. 3.3. Isolation of peripheral blood leukocytes (PBL) Peripheral blood ieukocytes were isolated by Ficoll Hypaque density gradient centrifugation as described by Boyum [8], washed three times with plain RPMI medium (200 × g, 10 min for each washing) and suspended in complete medium (RPMI 1640 medium containing 10~ (v/v) foetal calf serum). Cells were counted in a haemoeytometer and finally adjusted to the required cell concen!,ation in complete medium. 3. 4. Natural killer cell assay N K activity was studied in the monkey PBL using a standard chromium release assay [9] with human K562 tumour cells as target. Briefly, the assays were done in round bottomed, microtitre plates in a 15 h SICr release assay. PBL (10 x 106 ml -n, 100 t~i/wcll) from normal and infected monkeys were incubated with S]Cr-labelled target K562 cells (I × 104 cells in 100/H/well) at different effector: target (E:T) ratios for 15 h a t 3 7 ° C . At the end of the experiment 100 pl of the assay mixtures (TEST) were ~moved from each well and the SICr released was counted in an LKB Gamma counter, Spontaneous release (SR) of SICr was studied by incubating an equal volume of medium instead of effoctor PBL cells with the labelled target K562 cells. To estimate the maximum release (MR) of 5nCr, 1 × 104 labelled target

cells were suspended in 1.0 ml of distilled water, vortexed well and centrifuged at 500 × g for 10 rain. Radioactivity in 0.5 ml of the supernate was determined. The percentage specific lysis of target cells was calculated by the following formula: TEST cpm - SR cpm × 100 Percent lysis = MR cpm - SR cpm

4. RESULTS Five rhesus monkeys were infected with P. cynomolgi and the course of infection in each case was followed by determining the percentage parasit~emia in peripheral blood erythrocytes. Percentage of parasitaemia increased over a period of about sixteen days (the ascending phase) and peaked at an average of 11.5 =t: 5.27. Thereafter, over a period of about one week (the descending phase) the parasitaemia declined to an average of 0.08 + 0.I. This was followed by the recovery phase and during this phase parasites were eliminated from the peripheral blood stream and the animals were microscopically negative for parasites in blood. During each of the phases of infection the levels of anti K562 NK cell activity in the PBL was examined at threa E : T ratios (100, 50 and 25:1). The N K cell activity in an uninfected %*

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Fig, I. NK activity in peripheral blood derived from normal rhesus monkeysat diffet'entstages of p. eynomolgl infection. AP, ¢~:¢ndingphase,; DP, dcscondingphase; RP, recovery phase. Levelsof s~gnificanceweredeterminedby the Student's t-test. * P .~0.0l, * * P < 0.05.

123 Table 1 P. cynomolgi infection and differential leukocyte in rhesusmonkeys Day of Infection

Parasitaemia

Polymorph (Neutrophil)

Lymphc~,tes Moa.ocy tes (Percentageof tolal po~u alions)

Eosinophil

Basophil

9 11 12 13

0.06-1-0.04 0.6 =t-0.1 1.034-0.15 1.8 =1:0.2 3.5 +1.07 11.5 ±5.27 2.89±1.28 0.52+0.21 0.08±0.1 -re -ve

58=1:7.64 45+ 6.0 504- 5.5 304- 7.0 41± 3.6 444-10.45 224- 2.5 184- 3.0 22± 3 40_+ 5 504- 6

31-+ 3.05 48_+ 7.5 454-11.0 51+ 3.05 50+ 2.0 47± 9.0 63± 6.78 72-1- 7.50 67±10.0 584- 7.0 50± 0.0

1 l 2 ±1.5 l -+0.0

0 0 l 0

1 +0.0

0 0

72± 2 . 4 2

20:1:2.32

14

16 18 20 22 25 28 0(Notmalmonkeys)

-

4+1.5 3_+2.0 7±0.6 7+2.5 8±1.0 8+5.5 16+4.5 6:t=2.5 8--+2.0 3±1.5 3-*-1.0 4+1.05

t 4-0.7 0.84-0.45 1 +0 2 +1.5 0.5±0.5 1 2 +1.2

0 0 0 0 0 0.3:1:0.04

Rhesus monkeyswere inoculated with P. cyntmudgi(1 ×10s cells/monkey) on day O. Prepatent period was about 8 days. Results shownabovereprescmtmean±S.D., valuesof five infected normal monkeys. monkey was also determined in each experiment as a control. The combined results of these experiments, shown in Fig. 1, indicate that there was a significant fall in peripheral blood NK cell activity durin 8 the ascending phase (AP) of the infection. The descending phase (DP) of the infection was associated with normal or sfightly elevated N K cell activity (Fig. 1) whereas the levels of N K cell activity in control monkeys and monkeys in the post infection, recovery phase (RP) were not significantly different. The differential leukocyte counts in monkeys infected with P. cynomolgi showed that with the increase in parasitaemia, to day 16 there was a varying neutrophil count to day 16 accompanied by an increasing lymphocyte count to day 20 (Table 1). The lymphocyte peak value (72 + 7.5%) was observed in the declining phase of the infection. It is interesting to note that the increase in N K activity during this period correlated with d,e increase in lymphocyte counts. As with lymphocytes, the monocyte count increased to day 16-18 of the infection. The eosinophil and basophil counts remained low.

5. DISCUSSION Previously, a decline in the count of peripheral blood N K cells was reported in patients with P.

v i ~ malaria [6]. A follow-up study was difficult since the patients were not available for regular blood sampling. Moreover, the patients were on chemotherapy which could interfere with the pattern of changes in N K cell numbers during the course of infection. Due to its similarity with human P. vivax infection [10], the P. cynomolgi infection model of monkeys was used to assess the changes in N K cell activity during the course of infection. The results indicated a significant dedine in peripheral blood N K cell activity during the ascending phase (AP) of the infection as observed previously in P. vivax malarial patients [6]. However a decline in blood N K cells does not necessarily indicate suppression of the N K cell population. In turnout-hearing mice relocation of NK cells to the turnout mass may result in an apparent decrease in systemic NK cell numbers [11]. Interestingly, parasitized erythrocytes can competitively inhibit the lysis of K562 cells by the NK cells [12]. This finding may indicate that the infected erythrocytes may interact with the NK cells. If such an interaction can indeed take place, it is possible that the N K cells may relocate to liver and spleen containing infected cells thus resulting in an apparent decline in circulating NK cells. The increase in N K cell activity during the descending (DP) and recovery phases (RP) could result from the migration of N K cells back into the blood. In this context it should be noted that

124 the N K cells are capable of iysing several target cells and do not die after a single a n f o u n t e r with the target [13]. These suggestions a b o u t the migration of N K cells however are purely hypothetical at present a n d would require farther work for validation. The increase in N K activity during the decfining phase of infection seems to correlate with the m a x i m u m increase in the lymphocyte count. Interferon mediated N K call activation was reported in malarial patients [14]; this could contribute to the normalization of N K eel1 n u m b e r s d u r i n g the later phases of infection. The differential leukocyte cell counts o b t a i n e d in this s t u d y were reasonably similar to earlier observations in monkeys infected with 1". brasiIianum, P. coalneyi a n d P. falciparum, as shown b y the increase in blood lymphocyte a n d mono~ cyte count in infected monkeys |15]. Interestingly, the neutrophil c o u n t rose during the infection and fell between days 18 and 22, b u t tended to go up again (days 2 5 - 2 8 ) in a secondary surge. The relevance of these changes is not understood; the d a t a o n changes in the counts of other types of leukocytes m a y indicate an i m p o r t a n t role for these cells in i m m u n i t y to the malarial parasite. There is a need for additional work in this area.

ACKNOWLEDGEMENTS The technical assistance of Mrs. A. Sivaraman and A. Sharma a n d the help provided by the various publication sections of the Malaria Research Centre are gratefully acknowledged.

REFERENCES [ll Hailer, O, Hansson, M, Kiessling, It, and Wigzell, H. (1977) Role of non~eonvealienal natural killer cells in resistance against syngenie turnout cells in vivo. Nature 270, 609-611. 12] Solomon, J.B., Forbes~ M.G. and Solomon, G.ILA, (1985) A possible role for natural killer cells in providing prote¢-

lion against Plasmodium berghci in early stages of infeclion. lmmunoL Lett. 9, 352-359. [3] Eugui, E,M. and Allison, A.C, (1980) Differenfe~ in susceplibilitie~ of various mouse strains to haemopmtozoan infections. Possible correlation with natural killer activity. Parasite Immunol. 2, 277-292. [4] Wood, P.IL and Clark, I.A., (1982) Apparant irralevance of NK cells to resolution of infections with Baberia rmcroti and PImmodium vineki petteri in mice. Pai'asite ImmunoL 4, 319-327. [5] Chalenmpa, W., Auhasishtha, N.. Looaroesuwan, S. and Tharavanij, S. (1982) Natural Killer cells in peripheral blood of healthy individuals and patients with malaria. S.E. Asian J. Trop. Mud. Publ. Hl*h 13, 61-68. [6] Saxena, Q.B., Biswss, S. and Sharma, V.P. (1988)~ Status o! Natural Killer activity in the peripheral blood of P. vioax and P, falciparum patients. Indian J. Malariol. 25, 11-15. [7] Russel, P.F. (1963) Laboratory and field techniques, in Practical Malariology, 2nd Edn., p. 125. Oxford Univ. Press. [81 Boyum. A. (1968) Separation of leukoeytes from blood and bone masmw. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77-89. [9] Saxe~a, Q.B., Mezy, E. end Adler, W.H. (1980) The effect of alcohol consumption on human peripheral blood natural killer activity, int. J. Cancer 26, 413-417. [10l Bmce-ChwaU, LJ. (1984) The malarial parasite, in Essential Malariology, pp. 12-50, William Heinemann Medical Books Ltd, London. [11) Saxena, ILK., Saaena, Q.B. end Adler, W.H. (1983) Mechanism of loss of Natural Killer activity in P815 tumor bearing DBA/2 mice. Nat. lmmun. Cell Growth Regulation 3, 34-42. [12] Saxena, Q.B., Biswas, S. and Slmrma, V.P. (1989) Interaction of human natural killer cells with pIasmodium infected erythrocytes. Exp. Parasitol. 69, 300-302. [131 Timonen, T., Ortaldo, J.R. and Herberman, ILB. (1982) Analysis by a single cell cytoloxicity assay of natural ki~er ceil frequencies among unman large granular lymphooyles and the effect of IFN on their activity. J. lmmunol. 128. 2514-2518. [14] Ojo-Amaiz¢, E.A., Salimonu, L.$., Willlan~ A.LO., Akinwulere, O,A.O., Shaho, IL, Aim. G.V. and Wigzell, H. (1981) Positive correlation between degree o| parasitemia, interferon liters and natural killer activity in Plasmodium/alciparum infected children. J. Immunnl. 127, 22%-23~, [15] Aikawa, M., Suzuki, M. and Gntierz, Y. (1980). Pathology of Malaria. in Malaria, voL 2. (Krier, J.P., Ed.), pp. 47-102. Academic Press, London.