Clin. exp. Immunol. (1977) 27, 446-453.

Human polymorphonuclear leucocytes as mediators of antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells J. M. OLESKE, R. B. ASHMAN, S. KOHL, S. L. SHORE, S. E. STARR, PHYLLIS WOOD & A. J. NAHMIAS Department ofPediatrics, Emory University School ofMedicine, Georgia and Viral Immunology Branch, Centre for Disease Control, USPHS, Atlanta, Georgia, U.S.A.

(Received 1 September 1976)

SUMMARY

Human polymorphonuclear leucocytes (PML) were able to mediate antibody-dependent cellular cytotoxicity (ADCC) against target cells acutely infected with type 1 Herpes simplex virus. The reaction mediated by PML occurred more slowly and required higher concentrations of immune serum than that mediated by human mononuclear cells (MC). At the same ratio of effector cells to target cells, PML-mediated ADCC was less than MC-mediated ADCC. The observed relationship between the number of effector cells added, and the number of target cells lysed, showed that cytolysis mediated by both PML and MC was consistent with 'one hit' probability predictions. This suggested that target cell death resulted from an interaction with a single effector cell. The calculated frequency of effector cells in PML preparation was similar to that in MC, approximately 3.5%o. Preliminary examination of the nature of the effector cells suggested that they did not comprise a morphologically distinct subclass of PML. These experiments demonstrate a possible new role for PML in host defence against viral infections.

INTRODUCTION Although antibody-dependent cellular cytotoxicity (ADCC) has been described in a variety of experimental systems, many investigators have chosen to use mononuclear leucocytes as effector cells (reviewed by Perlmann, Perlmann & Wigzell, 1972). Nevertheless, the reaction can also be mediated by such diverse cell types as polymorphonuclear leucocytes (PML) (Gale & Zighelboim, 1974), monocytes (Holm & Hummarstrom, 1973) and macrophages (Holm, 1972), as well as by some reticular cell lines (Walker & Demus, 1975; Ralph, Prichard & Cohen, 1975). The ADCC reaction has been shown to require direct contact between the target cell and the effector cell, which occurs through the binding of effector cell surface Fc receptors to the Fc epitopes of target cell-bound antibody, usually of the IgG class (MacLennan, 1972; Larsson & Perlmann, 1972; Trinchieri et al., 1975). The actual mechanism by which the lethal hit is mounted, however, is still incompletely understood. In addition, there is no evidence as yet that the various effector cell types are equally efficient mediators of ADCC, or that the characteristics of the cytotoxic reaction are the same when different effector cell types are used. Mononuclear cell (MC)-mediated ADCC against herpes simplex virus (HSV)-infected cells has been described in both murine (Rager-Zisman & Bloom, 1974; Ramshaw, 1975) and human (Shore et al., 1974; Russell, Percy & Kovithavongs, 1975; Shore et al., 1976a) experimental systems. This report describes for the first time ADCC against HSV-infected target cells mediated by human PML and compares the various parameters of this reaction with those of MC-mediated ADCC. Correspondence: Dr J. M. Oleske, Department of Pediatrics, New Jersey College of Medicine, Newark, New Jersey 07 100.

446

Human PML-mediated ADCC to HSV-infected cells

447

MATERIALS AND METHODS Cell line. Chang liver (CL) cells were grown in 6-ounce prescription bottles in Eagle's minimum essential medium (MEM) containing penicillin, streptomycin and fungizone, supplemented with 10% foetal calf serum (FCS). Sera. Serum samples from healthy donors were tested for antibodies to HSV-1 by microneutralization (Nahmias et al., 1968). Pools were made of serum from five positive and five negative donors, inactivated at 560C for 30 min, and stored at - 200C. Effector cells. Healthy donors were classified as being either HSV-positive or negative on the basis of the presence or absence of serum antibodies to HSV. Blood was drawn into heparinized syringes containing 1 ml of 6% Dextran (Pharmacia, Piscataway, New Jersey) per 10 ml of blood, and the cells were allowed to sediment at 370C for 45 min. The leucocyte-rich plasma was recovered, layered over a Ficoll-Hypaque gradient, and centrifuged at 400 g for 45 min (Boyum, 1968). MC were recovered from the interface, and PML from the cell pellet. The cells were washed three times in Hanks's balanced salt solution, and once with MEM containing 10% inactivated FCS. Total and differential cell counts were then performed, and the cells diluted to the appropriate effector to target cell (E:T) ratio in MEM containing 10% inactivated FCS. ADCC assay. The technique used was essentially the same as that previously described (Shore et al., 1974, 1976a). Confluent monolayers of CL cells were inoculated with HSV-1 (HE strain) at a multiplicity of one to two plaque-forming units per cell. When the cytopathic effect involved about 50% of the cells, usually 24 hr after inoculation, the infected cells were removed using trypsin-EDTA (Grand Island Biological Company, New York) and washed twice with MEM containing 2% inactivated FCS. An aliquot of 6 x 106 cells was incubated with 100 ,uCi "'Cr (sodium chromate, New England Nuclear, Boston, Massachusetts), in a final volume of 1 ml for 90 min at 37C. The labelled cells were then washed four times and adjusted to a concentration of 5 x 104 cells/ml in MEM with 10% inactivated FCS. Uninfected cells were prepared in the same manner. Four-tenths millilitre of effector cells and 0-2 ml of human serum were added to 0 4 ml of target cells in 12 x 75 mm plastic tissue culture tubes (Falcon Plastics no. 2054), and gently agitated. The tubes were then incubated at 370C in 5% CO2 in humidified air. At the end of the incubation period the tubes were shaken, and centrifuged at 300 g for 15 min to pellet the cellular material. Release of 5 1Cr from the cells was determined by withdrawing the top 0 5 ml of the 1 0 ml reaction volume, and determined the radioactivity in each fraction by counting in a Beckman Biogamma II gamma counter. Percent release of 51Cr was calculated by the formula: 2A R =- x 100, A+B where R = per cent release; A = ct/min top 0 5 ml and B = ct/min, bottom 0 5 ml. Percent specific cytotoxicity was defined as follows: CT= C-D where CT = per cent specific cytotoxicity; C = per cent release from target cells exposed to effector cells and human serum; and D = per cent release from target cells exposed to effector cells alone. All experiments were performed in triplicate. The standard error of CT was calculated from the standard deviations of C and D, using a computer programme developed by Emory University Computer Center.

RESULTS

Characterization of PML as effector cells Smears were made of cells from both PML and MC preparations in most experiments, and differential counts were performed. The relative proportion of the different cell types present in the PML and MC preparations are shown in Table 1. Although MC contamination of the PML preparations did not exceed 3% in these experiments, it was important to determine the levels of cytotoxic activity that might be attributable to the small number of contaminating MC. Accordingly, control ADCC cultures were set up with each experiment, using MC at the E :T ratio which corresponded to the percentage of MC contamination of the PML preparation used that day. For example, if the PML preparation used in a given experiment was contaminated with 2% MC and was being used at an E:T ratio of 100:1, the MC ADCC control cultures consisted of target cells, antibody, and MC at an E :T ratio of 2:1. In no experiment did the contaminating MC make a major contribution to the observed cytotoxicity of PML preparations (e.g. Figs 1 and 2). PML-mediated cytotoxicity was not influenced by the donor immune status to HSV.

Controls for specificity of cytotoxicity Spontaneous 5tCr release from the target cells after 16-hr incubation was similar to that described previously (Shore et al., 1976a): between 15 and 22% for HSV-1 infected CL cells, and between 25

J. M. Oleske et al.

448

TABLE 1. Percentage of various leucocyte types in the preparations of PML and MC used as effector cells in ADCC Leucocyte morphology*

Cell Preparation PMLt

MCI

PML

MC

Eosinophil

Juvenile PML

84-1+1-9 69+10

10+03 931+10

6-0+13 00

91+1-3 00

* Based on Wright's stained smears. t Pellet from Ficoll-Hypaque density gradient separation of dextran-sedimented leucocytes. Mean+ s.e.m. of thirteen experiments. I Interface cells from Ficoll-Hypaque density gradient separation of dextran sedimented leucocytes. Mean+ s.e.m. of eleven experiments.

TABLE 2. Specific lysis of HSV-1 infected target cells by human PML and MC after 48 hr in culture,* compared with freshly prepared effector cells

Time in culture 48 hr

0 hr

Cytotoxicity (%)

ViabilityT

Effector cell

(%)

Cytotoxicity (0)

Viability (%)

PML (50:1)t MC (50:1) MC (2:1)¶T

145+54§ 39 2+ 3 9 3 1+1 6

98 95 95

-09+07 14 4+ 1.0 3 1+3 1

0 75 75

* Cells were cultured at a density of 2 5 x 105/ml in 2-ml cultures in MEM supplemented with 10% heat-inactivated FCS before use in standard 16 hr ADCC assay. t E :T ratio is shown in parentheses. t Viability was determined by exclusion of Erythrocin B dye. § Mean+ s.e.m. of triplicate determinations. T Control for MC contamination of PML.

TABLE 3. Specific lysis by human PML and MC of HSV-1infected target cells pre-incubated with HSV antibody-positive serum.* The results are compared with those from a routine assay in which antibody and effector cells were added simul-

taneously

Effector cell PML (100:l)t

MC(50:1)

Target cells pre-incubated with antibody

Routine assay of positive sera

9 5 + 2 4t 237+12

13 4+ 2 4 330+08

* 5 x 106 target cells were incubated with 1:10 final dilution of sera for 30 min at room temperature then washed four times with cold MEM supplemented with twenty-five heat-inactivated FCS.

t E :T ratio is shown in parentheses. $ Mean + s.e.m. of triplicate determinations.

Human PML-mediated ADCC to HSV-injected cells

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and 35% for uninfected cells. Serum controls were run in each experiment; however, because neither HSV-positive, nor HSV-negative serum alone caused significant 5tCr release from either HSV-1 infected or uninfected cells, these data are omitted. MC, in the absence of antibody, caused a slight increase, typically less than 10%, in 51Cr release from both infected and uninfected cells. In contrast PML alone did not cause any nonspecific 51Cr release. Neither MC nor PML caused specific ADCC against uninfected CL cells in the presence of HSV-1 positive or negative serum, so these data are also omitted.

Viability of PML and MC used as effector cells in ADCC Initial viability of both PML and MC, determined by exclusion of Erythrocin B dye, was greater than 98%. After the typical 16 hr incubation period, 80% of the PML remained viable. Thereafter, the viability decreased rapidly, and all PML were dead after 48 hr in culture. On the other hand, 75% of MC remained viable after 48 hr under the same culture conditions. The differential survival of 70

60 50

0~

40

T

-

20-

10 _ 0

\ 1:10

1:30

1:100 1:300 1:1000 1:3000 Antibody dilution

FIG. 1. Specific lysis of HSV-1 infected CL cells by human PML or MC in the presence of varying dilutions of serum antibody to HSV. Each point represents the mean+ s.e.m. of five experiments. ( * *) PML at E:T ratio of 100:1; (A- --A) MC at E:T ratio of 50:1; (A-- ) control for MC contamination of PML, E:T ratio < 3:1.

PML and MC in culture permitted us definitively to exclude the possibility that the observed cytotoxicity could be due to the selective enrichment of the PML with 'K' or 'null' cells. ADCC effector cell activity of freshly prepared PML and MC and those of PML and MC after 48 hr in culture are shown in Table 2. After 48 hr in culture, all the PML were dead, and their cytotoxic effector cell activity was completely lost. The MC, on the other hand, showed only a 20% loss of cell viability; effector cell activity was still easily demonstrable, although it was only about one-third of its pre-culture value.

Failure to demonstrate ADCC by preincubation of effector cells with antibody In order to determine whether the observed cytotoxicity could be due to 'arming' of the PML by serum antibody, 5 x 106 PML and MC were incubated with a 1:10 dilution of either HSV antibodypositive or HSV antibody-negative serum for 30 min at 370C. The cells were then washed four times to remove excess antibody and tested for cytotoxicity against HSV-1 infected targets at E:T ratios of 100:1 (PML) and 50:1 (MC). In no instance did the pretreatment of either PML or MC with either antibody-positive or antibody-negative serum render them more cytotoxic to the virus-infected target cells than untreated PML or MC.

J. M. Oleske et al. Induction ofADCC by sequential addition ofantibody and effector cells

450

Since the cytotoxic serum factor did not appear to bind to the effector cells, incubation of the target cells with HSV-antibody positive serum should render them susceptible to lysis by PML, as has been previously demonstrated for MC (Shore et al., 1976a). Table 3 depicts the results of a typical experiment in which HSV-1 infected CL cells were incubated with either HSV antibody-positive or HSV antibodynegative serum, and cultured for 16 hr with either PML or MC at E:T ratios of 100:1 and 50:1 respectively. These data are compared with the results of a routine ADCC assay, in which immune serum and effector cells were added simultaneously. Both PML and MC were capable of lysing the antibodytreated target cells, although the levels of specific cytotoxicity were somewhat reduced when compared with the routine assay. Antibody requirements for PML-mediated ADCC The effect of varying concentrations of HSV immune serum on PML-mediated and MC-mediated ADCC was determined using serum dilutions of 1:10-1:3000. Cytotoxicity was measured after a 16-hr incubation period. The requirements of PML and MC for antibody in the ADCC reaction were strikingly different (Fig. 1). Optimal PML-mediated ADCC required serum dilutions of 1:10 to 1:100. 55-

50 45

T

40

10

/

35 2 30

I

25-

020-

o

S

2

4

8 Incubation time (hr)

16

FIG. 2. Relation of incubation time to specific lysis of HSV-1 infected CL cells by human PML or MC in the ) presence of serum antibody to HSV. Each point represents the mean+ s.e.m. of four experiments. (0* PML at E:T ratio of 100:1; ( A- - - A) MC at E:T ratio of 50:1; (*--- ) control for MC contamination of PML, E:T ratio < 3:1.

Cytotoxicity at higher dilutions of serum decreased steadily and was undetectable at a serum dilution of 1:1000. In contrast, significant MC-mediated ADCC was demonstrable at the highest serum dilution employed (1:3000). Kinetics of PML-mediated and MC-mediated ADCC Cultures were established using PML at an E:T ratio of 100:1 and MC at 50:1, and cytotoxicity was measured at 2, 4, 8, and 16 hr. There was a considerable difference between PML-mediated ADCC and MC-mediated ADCC in both the rapidity with which cytotoxicity could be detected, and in the shape of the reaction curve (Fig. 2). PML-mediated ADCC was first detected, at very low levels, 4 hr

Human PML-mediated ADCC to HSV-infected cells

451

after the cells were established in culture, and increased linearly through 16 hr. The reaction reached completion between 16 and 24 hr. In contrast, the MC-mediated reaction was more rapid: high levels of MC-mediated ADCC were demonstrated within 2 hr, and maximum specific cytotoxicity was obtained within 8 hr.

Effect of increasing E :T ratios The increase in cytotoxicity obtained by adding increasing numbers of effector cells to a constant number of target cells was determined us ng PML and MC at E:T ratios of 1:1, 5:1, 10:1, 50:1 and 100:1. The cultures were harvested after 16 hr incubation. When the percent cytotoxicity was plotted against the E:T ratio, the curves for both PML and MC were strikingly similar (Fig. 3), although the PML-mediated ADCC reaction was uniformly lower than the MC-mediated reaction. Both curves were concave to the abscissa, which suggested that PML, like MC (Ziegler & Henney 1975), only require 'one hit' for antibody-mediated cytolysis to occur. 65 60

-

53 _Ii- -45

40

I

x

° 35 -I

~30 25

20 15

5A 1:1

5:!

10:1

50:1 Effector to target cell ratio

1001

FIG. 3. Specific lysis of HSV-1 infected CL cells by human PML or MC at different E :T ratios in the presence of serum antibody to HSV. Each point represents the mean+ s.e.m. of seven experiments in the case of PML ( * *), five experiments in the case of MC (--- A).

When the number of target cells specifically lysed was fitted to a Poisson probability distribution and plotted on a log/log scale against the number of effector cells added (Ziegler & Henney, 1975), the slope for the PML was not significantly different from 10 (mean+ s.e.m. of four experiments = 0 89+ 0.10 s.e.m.). This relationship was also determined for the MC-mediated reaction in the same four experiments and the mean slope found to be 0 94+0 05 s.e.m. The frequency of effector cells in the MC preparation was calculated to be 3-56+ 0 58% s.e.m.; the calculated frequency of effector cells in the PML preparation was similar, but highly variable, with a mean of 3-65 + 2-5 1% s.e.m. The relatively low frequency of cytotoxic effector cells in the PML preparation suggested that the effector cells might be a morphologically distinct subpopulation, such as the eosinophil or the juvenile forms of the PML. If this were the case, there should have been a direct correlation between the proportion of the particular cell type in the PML and the level of cytotoxicity. No such correlation could be demonstrated.

452

J. M. Oleske et al.

DISCUSSION These results demonstrate that human PML are capable of mediating ADCC against allogeneic target cells acutely infected with HSV. PML-mediated ADCC requires longer incubation time for the expression of cytolysis and a higher concentration of HSV-antibody positive human serum than does MC-mediated ADCC. In addition, the PML effector cell activity is completely abrogated after 48 hr in culture, whereas about one-third of the MC effector cell activity is retained under similar culture conditions. The different characteristics of the PML-mediated reaction, as compared to the MCmediated reaction, offer further evidence that the effector cell activity of the PML preparations is not due to the low level of contamination of PML preparations with MC. The effector cells in the two types of ADCC reactions, therefore, are clearly different. Neither the PML effector nor the MC effector cell can be directly 'armed' by preincubation with a source of HSV antibody; however, lysis can be induced after preincubation of the target cells with specific antibody. This is further evidence that effector cell activity is induced by the formation of antigen-antibody complexes on the surface of the target cell (MacLennan, 1972; Perlmann et al., 1972). The antibody which is required for MC-mediated ADCC against HSV infected target cells has been found to be of the IgG class (Shore et al., 1976a), and it is also known that the Fc receptors on human mononuclear effector cells react with all four subclasses of human IgG (MacLennan, 1972). Although it is likely that PML-mediated ADCC also requires an antibody of the IgG class, the PML may only recognize some of the IgG subclasses, a situation which would result in an apparent need for a higher concentration of HSV immune serum. An alternative interpretation is that PML may have an absolute requirement for a greater total density of target cell-bound antibody than do MC for the expression of ADCC. Most ADCC systems studied to date have used MC as effector cells, and there is now considerable evidence that the mononuclear effector cells in human blood are lymphocytes which bear Fc receptors on their surface, but which are neither classical T cells nor B cells (Wisloff, Fr0land & Michaelsen, 1974; Calder et al., 1974; Shore et al., 1976b). These 'K' cells are capable of lysing HSV-infected target cells within 2 hr (Shore et al., 1974; Shore et al., 1976a). In contrast, 4-8 hr are required for PML to lyse a significant number of sensitized HSV-infected targets. The slower rate of PML-mediated, as compared to MC-mediated ADCC, may reflect either a difference in the rapidity with which the effector cells bind to the target cells, or a difference in the time after binding at which cell lysis is effected. The time kinetics of PML-mediated ADCC in the HSV experimental systems are in contrast to the report of PML-mediated cytotoxicity to xenogeneic target cells, in which appreciable cytotoxicity was noted after two hours incubation (Gale & Zighelboim, 1974). Whether this difference in reaction velocity reflects a difference in the susceptibility to lysis of different target cell types, or a difference in the subpopulations of PML which mediate ADCC in the two experimental systems, remains to be determined. The lysis of target cells by human peripheral blood lymphocytes has been shown to follow from a single interaction between effector and target cell (Ziegler & Henney, 1975), and the kinetics of target cell destruction by human PML are also consistent with 'one hit' probability predictions. In these experiments, the frequency of effector cells in the MC preparation was estimated to be approximately 3-5°/ a value very close to the 4% reported by Ziegler & Henney (1975). The estimated frequency of effector cells in the PML preparation was 3.6%; however, the variability between the four individual estimates for PML was very large and suggested that the population of cytotoxic effector cells varied markedly between individuals. This was consistent with the observation that PML preparations from certain individuals showed equal or superior reactivity to that of their MC. The variability in the proportion of eosinophils and juvenile forms in our PML preparations led us to examine the possibility that one of these subpopulations might comprise the majority of the effector cells. Both eosinophils (Butterworth et al., 1975; Oleske, Ashman & Nahmias, unpublished data) and neutrophils and intermediary granulocytes (Penfold, Greenberg & Roitt, 1976) have been shown to be capable of acting as effector cells in ADCC; however, in the HSV experimental system, we have not been able to identify a single, morphologically distinct, type of PML which appears to be the predominant effector cell in PML-mediated ADCC.

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453

The role of PML-mediated ADCC in vivo remains to be determined. It has long been known that large numbers of PML are present in herpetic lesions, and recent studies have suggested a possible mechanism for the accumulation of PML in recurrent lesions. Snyderman, Wohlenberg & Notkins (1972) have shown that HSV absorbed to the surface of cells will interact with antibody in the presence of complement to generate a factor chemotactic for PML. This reaction takes place before viral penetration into the cell, but at this stage of infection complement-dependent antibody cytolysis cannot be demonstrated. PML-mediated ADCC thus provides a mechanism by which the PML attracted to the lesion might restrict viral proliferation by lysing infected cells before the occurrence of viral cell-to-cell spread. It will be important in future experiments to ascertain the earliest stage in the infectious cycle at which PML-mediated ADCC can occur. This work was supported in part by National Cancer Institute fellowships 1 F32 CA05232, and 1 F22 CA00577-01, by contract CP 43393 from the NCI, by grant 5 RO1-DE 03924 from the National Institute of Dental Research and by Training programme Al 00447-04 of National Institute of Allergy and Infectious Disease, National Institutes of Health, Public Health Service. REFERENCES

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