Early enhancement followed by suppression of natural killer cell activity during murine malarial infections

Immunology Letters, 2 (1981 ) 209-212 © l'21sevier/North-HollandBiomedical Press

E A R L Y E N H A N C E M E N T F O L L O W E D BY S U P P R E S S I O N O F N A T U R A L K I L L E R C E L L ACTIVITY DURING MURINE MALARIAL INFECTIONS Kenneth W. ttUNTER, Jr., Thomas M. FOLKS, Peter C. SAYLES and G. Thomas STRICKLAND Departments of Pediatrics and Medicine, Un(fbrmed Services University School of Medicine; and The Department of Immunology, Naval Medical Research Institute, Bethesda, MD, 20014, U.S.A. (Received 30 June 1980) (Modified version received 16 September 1980) (Accepted 17 September 1980)

1. Introduction

parasitemia levels were determined from Giemsastained thin films of tail blood.

Malarial infections can modulate immune reactivity through a generalized suppression of humoral and cellular components of the host immune response. This is exemplified by impaired antibody responses to certain antigens [ 1-3], decreased mitogen responsiveness [4,3], and delayed graft rejection [6]. Furthermore, concomitant infection with malaria leads to defective immunity to other organisms [1,7]. On the other hand, malarial infections have not been shown to potentiate specific components of the immune response. In this paper, we describe an early enhancement of splenic natural killer (NK) cell activity which is followed by marked suppression later in the course ofP. yoelii infections in AKR/J mice. This early potentiation of NK activity correlated with a transient early rise in serum interferon levels. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) and the responses of thymus-derived (T) and bone marrow-derived (B) lymphocytes to mitogens were supp.ressed throughout the course of infection.

2.2. N K and ADCC assays Cultured YAC-1 tumor cells or fresh chicken red blood cells (CRBC) were incubated for 1 h at 37°C with 100/ICi of Na2 SlCrO4 (Amersham Searle, Arlington Heights, IL), washed and added to round bottom microtiter plates (Linbro Scientific, Hamden, CN) at 2.0 × 104 cells per well. Ammonium chloridetreated spleen cells were added to the chromiumlabeled target cells at ratios of 50:1 (YAC-1) or 25 : ! (CRBC). For the ADCC assay, rabbit anti-CRBC (Cappel Laboratories, Cochranville, PA) was added with the CRBC target cells at a 1:20,000 final dilution. For both cytotoxic assays, the total volume per well was 200 pl. Plates were incubated at 37°C for 4 h, then centrifuged and 100/al of supernatant was removed and counted in a gamma-counter. The per cent cytotoxicity was calculated as: cpm of cytotoxic chromium release-cpm of spontaneous chromium release/ cpm from total lysis (freeze-thaw) X 100.

2. Materials and methods

2.3. Interferon assay The ability of serum to inhibit the cytopathic effect of vesicular stomatitis virus was used to assess interferon activity. The assay was performed by Biofluids, Rockville, MD.

2.1. Mice and parasites Six-week-old female AKR/J mice (Jackson Laboratories, Bar Harbor, ME) were challenged intraperitoneally with 1.0 X 10 6 erythrocytes infected with the avirulent 17 X strain ofPlasmodium yoelii, and blood

2.4. Mitogen-induced proliferation Aliquots of 5.0 × lO s spleen cells from infected and non-infected mice were cultured in flat bottom

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microtiter plates (Dynatech Laboratories, Alexandria, VA). The total volume per well was 200/al, which consisted o f cells in RPM1 1640 medium (Microbiological Associates, Rockville, MD) supplemented with 2 mM L-glutamine; 10% fetal bovine serum (North American Biologicals, Miami, FL), and 50 l.U./ml penicillin and 50/xg/ml streptomycin. Concanavalin A (Con A) (Calbiochem, San Diego, CA) and bacterial lipopolysaccharide (LPS) (Difco Laboratories, Detroit, MI, from E. coli 0127:B8) were tested .in optimal stimulatory concentrations of 1 #g/ml and 10/~g/ml respectively. After 48 h of culture with mitogens in a humidified 5% CO2 atmosphere, 1/aCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to each well. The cultures were harvested 16 h later using an automated cell harvester, and the [3H]thymidine-uptake of the spleen cells was measured in a beta-scintillation counter.

3. Results

At different times after P. yoelii challenge we examined the ability of non-sensitized spleen cells to kill allogeneic tumor cell targets (NK activity). On day 3 after challenge the malaria-infected mice

demonstrated a greater than 2-fold potentiation of splenic NK activity as compared to non-infected mice (Table 1). NK activity was still augmented on day 8, but to a lesser degree than on day 3. By day 14 of infection, near the point of peak parasitemia levels, NK activity became markedly suppressed and remained suppressed for as long as 34 days after P. yoelii challenge, even following complete elimination of circulating parasitized cells. Interferon is the most widely accepted potentiator of NK activity, and substances or conditions which induce the production of interferon also increase NK activity [8,9]. Therefore, in order to determine whether malarial infection induced an elevation in serum interferon levels, a separate experiment was performed in which A K R / J mice were infected with P. yoelii, then groups were sacrificed daily and their serum collected and analyzed for interferon. As coinpared to non-infected mice, malaria-infected mice demonstrated a 35-fold increase in serum interferon on day 1 after challenge, and a 10-fold increase on day 2 (Table 2). By day 3 post-infection serum interferon had returned to control levels. The influence of malarial infection on cytotoxicity mediated by the NK cell was then compared to ADCC against chicken erythrocytes, the effector cell of

Table 1 Splenic NK activity and ADCC in AKR/J mice at various times following challenge with P. yoelii DaYs post-infection

% Parasitemia

% Cytotoxicity (control:infected)

% of Control

<1 5 24 <1 0

13 + 10:28 -+4* 9+- 2:16+- 2* 18+ 4: 3-+0.5" 19+- 5: 5-+ 1" 13-+ 2: 5-+ 2*

215 178 17 26 38

3

<1

8 14

7 32

51 +- 8:45 -+ 5 32 +- 2:24 -+4* 44 -+5:23 +- 2*

NK activity 3 8 14 21 34 ADCC 88 75 52

The data are pooled from several experiments with 4 - 6 mice per group. The % cytotoxicity is expressed as the mean +-one S. D. of the mean. An asterisk denotes control means which are statistically different from infected means (P < 0.05, Students t-test). ~' 210

Table 2 Interferon levels in the sera of AKR/J mice following infection with P. yoelii Days post-infection Non-infected control

Units of interferon/ml (mean)

l 100

[ ] CONA ~LPS

9O 8O

20 6O

Infected 1 2 3-7

708 200 20

Four mice were sacrificed on the days indicated and serum collected from axillary blood was tested for levels of interferon as described in section 2.

which is the macrophage [10]. As compared with cytotoxic responses of non-infected mice, malariainfected mice demonstrated suppression of ADCC on days 3, 8 and 14 post-infection (Table 1), and the suppression was most marked on day 14 at the time of peak parasitemia levels. Thus it appeared that, unlike its effects on NK activity, malarial infection did not provide an early enhancement of macrophagemediated cytotoxicity. In a parallel series of experiments we examined another parameter of immune function - the spleen cell response to mitogens. The in vitro blastogenic responses to optimal concentrations of the T-cell mitogen, Con A, and the B-cell mitogen, LPS, were studied at various times after P. yoelii infection. As compared to the early enhancement of NK activity, the Con A response was more than 80% suppressed 2 days post-infection (Fig. 1). With minor variability, this level of suppression was maintained through 14 days after challenge. The LPS response was more gradually suppressed, progressing from 30% suppression on day 2 to 80% suppression on day 14. By 22 days after challenge, when blood films were negative for parasites, both Con A and LPS responses were completely abrogated.

4. Discussion These studies demonstrate a phenomenon not previously described during malarial infections; early enhancement of NK activity followed later by suppression. The importance to host resistance of the

i '° 50

40

30 20 tO

1

2

3

6 8 10 DAYPOSTINFEC~ON

12

14

22

Fig. 1. Per cent suppression of the mean proliferative response of spleen cells from infected mice relative to the mean proliferative response of non-infected mice after stimulation with Con A or LPS. The data are pooled from several experiments with at least 4 mice per point. early enhancement of NK activity cannot be assessed on the basis of the present results. Although Allison et al. [11] have recently described a protective effect of NK cells against blood protozoa, in a few limited experiments we have been unable to detect significant NK activity against parasitized erythrocyte targets. However, we have recently found a positive correlation between the level of NK activity in inbred strains of mice and resistance to malarial infection (Hunter et al., unpublished data). The early stimulation of interferon production in the present study compared favorably with that observed during P. berghei infections in mice [12], and may have some relevance to host resistance since there is evidence for a protective effect of interferon in rodent malaria [ 13,14]. If interferon is itself antiparasitic, mice which are stimulated to produce high levels of interferon or mice which are genetically high interferon producers would be resistant to the parasite. On the other hand, interferon may be involved in protection against malaria only indirectly through its effect on NK activity. After the first two weeks of malarial infection, NK activity became markedly suppressed. Although decreasing levels of interferon might explain the less significant enhancement of NK activity on day 8, the mechanism of the subsequent frank suppression 211

remains obscure. Unlike NK activity, ADCC against heterologous erythrocytes showed no early enhancement, but rather a progressive suppression. Again, the mechanism of suppression is unknown, but there is evidence that induction of interferon with Corynebacterium parvurn suppresses cytotoxicity mediated by the macrophage [10]. We also showed a rapid and progressive suppression of mitogen responses similar to that observed by others [4,5]. There is recent evidence that interferon can markedly suppress mitogen responses [15], and this could account for the early suppression of blastogenesis observed in our study. However, since the elevated interferon levels rapidly disappeared and the suppression of blastogenesis continued, it is unlikely that interferon alone can explain the suppression. In conclusion, we have shown that P. yoelli infection in AKR/J mice causes an early enhancement of cytotoxicity mediated by the NK cell, which may be correlated with a transient early stimulation of interferon. These findings may be relevant to host resistance to malarial infection since anti-malarial effects of both NK cells and interferon have been reported by others [ 11,13,14]. Although NK activity eventually became suppressed, the kinetics differed markedly from the early suppression of blastogenesis and ADCC. Further studies of the enhancement-suppression phenomenon and the relationship between interferon and NK activity should provide new insight into the mechanisms of host resistance to malarial infection.

Acknowledgements This work was supported by Uniformed Services University of the Health Sciences, Protocol R08305, and the Naval Medical Research and Development Command, Work Unit No. ZF58.524.013.1029. The opinions and assertions contained herein are the private ones of the authors and are not to be construed

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as official or reflecting the views of the Department of Defense, the U.S. Navy Department, or the Naval Service at large. The experiments reported herein were conducted according to the principles set forth in the 'Guide for the Care and Use of Laboratory Animals,' Institute of Laboratory Animal Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.

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