Induction of natural killer cells by herpes-simplex virus type 2 in resistant and sensitive inbred mouse strains

Immunobiol., vol. 158, pp. 369-379 (1981)

Immunology Department, Sandoz Forschungsinstitut, Vienna, Austria

Induction of Natural Killer Cells by Herpes-Simplex Virus Type 2 in Resistant and Sensitive Inbred Mouse Strains D. ARMERDING and H. ROSSITER Received September 9, 1980 . Accepted November 11, 1980

Abstract Infection of mice with herpes simplex virus type 2 (HSV 2) stimulated natural killer (NK) cells in a variety of inbred mouse strains including athymic nude mice. Essentially all mouse strains tested exhibited high NK activity on day four after virus inoculation. Assayed 24 hours after infection, SWR/J, AKR/J, SJLlJ and C57BilI0J mice were low or negative for these non­ virus-specific cytotoxic responses . Whereas the first two mouse strains were most sensitive to the lethal effects of HSV 2, the latter two were highly resistant. Three lines with intermediate susceptibility and three highly resistant strains were all efficient with regard to early NK -cell induction.

Introduction Natural resistance to virus infections in mice has been demonstrated by many authors to be genetically determined (1-10). In particular, the work of C. LOPEZ (5) documented a polygenic trait of four or more genes for the natural defence against herpes simplex virus type 1. Several non-antigen­ specific cellular functions have been suggested by this author (5) and by others (1-25) to be responsible for this type of inherited resistance. Mac­ rophages have been demonstrated to represent an important barrier against HSV (11-16) and other virus infections (reviewed in 17-19, see also 2, 4, 6). Interferon has been shown to yield another effective defence system (9, 20, 21). Natural killer (NK) cells have gained major interest over the recent years, especially in association with interferon which is able to activate this cell population (reviewed in 22-24, see also 25). Natural killer cells are induced by various viruses as shown by several authors (24-27) and by us l ) for HSV 2. They can express preference for virus-infected target cells (21, 24, 25,1» and probably leave non-infected 1) ARMERDlNG, D., M. SIMON, U. HAMMERLlNG, G. J. HAMMER LlNG , and H. ROSSITER. 1980. Function, target cell preference and cell surface characteristics of herpes simplex virus type 2 induced non-antigen specific killer cells. Immunobiol., this issue.

Abbreviations: HSV: Herpes simplex, virus; H-2: Major histocompatibility gene locus of the mouse; i.p.: intraperitoneal; NK: Natural killer; PEe: peritoneal exudate cell.

370 . D. ARMERDING and H. ROSSITER

cells intact under physiological in vivo conditions if interferon is present at the same time (24, 28). Thus a correlation between natural resistance to a particular virus species and its capability to induce NK cells might be found in some mouse strains. The aim of this study was to compare the resistance status to HSV 2 and in vivo non-virus-specific cytotoxic responses induced by the same virus in several inbred mouse lines. We will demonstrate in great detail elsewhere!) that after intraperitoneal injection of live HSV 2 preparations a non­ antigen-specific cytotoxic cell population is activated in the peritoneum. Cytotoxic responses exclusively involve cells which fulfill the criteria of NK cells (22-24) as verified by analysis of their cell-surface antigens, their mode of action, and their selectivity for particular target cells. Such responses develop within 24 hours and last up to eight days. During this time-period, no other cell classes expressing cytotoxicity could be detected in our system. The results reported here document that some mouse strains are ineffec­ tive NK-cell producers, and others exhibit fast and high responses. Two out of four low NK strains are resistant to HSV 2, the others highly sensitive. Thus NK cell induction is not a prerequisite for natural anti-viral protec­ tion. However, it could be that HSV 2-susceptible strains are sensitive because they fail efficiently to activate NK cells.

Materials and Methods Animals AKRI], CBAI], C3H/He], C57BI!Ks], C57BI!6], C57B1!10J, DBA/1], DBA/2], SWRI], S]LI] mice were obtained from Jackson Laboratories (Bar Harbour, Me., U.S.A.). Balb/c/ABom, C3H/TifBom, nu/nu Balb/c mice were derived from Gl. Bomholtgard Ltd. (Ry, Denmark). C57BRc/Han and C3H/HeHan mice were purchased from Zentralinstitut fiir Versuchstierzucht (Hanover, W. Germany). Supplier for C57Bl!10ScSn Ola, C57Bl!10ScSn nu/Ola and CBA/Ca nu/Ola mice was Olac, Shaws Farm (Blackthorn, England). C3H/Bi Mai mice were obtained from Microbiological Associates (Walkersville, Md., U.S.A.). Only male mice 12 weeks or older were used.

eelllines Exponentially growing L929 fibroblasts, PS15 mastocytoma cells, 3T6 (Swiss mouse derived) fibroblasts and Neuro 2A neuroblastoma cells maintained in tissue culture were used as target cells in the cytotoxicity assays. Cell lines were screened regularly for presence of mycoplasms and discarded if contaminated.

Viruses Herpes simplex virus type 2 strain G - grown on BHK cells - and strain 72 - grown on primary rabbit kidney cells - have been used. Both strains yielded similar results with regard to lethality and induction of cytotoxic effector cells in different mouse strains. Most experiments

NK Cells in HSV -resistant and -sensitive Mice . 371 presented here were done with strain G which grew to higher titers (10 7-108 PFU/ ml) than strain 72. HSV 2 was titrated by plaque formation on Vero cells under fluid overlay containing 0.1 % of rabbit hyperimmune serum against HSV 2. Virus preparations used were free of contaminants, such as mycoplasms. No attempt was made to purify the virus since virus-free control preparations did not induce any cytotoxic responses in all mice tested. Virus was stored in aliquots at -70°C and frozen and thawed only once.

Effector celJs Mice were sensitized with 0.2 ml HSV 2 at concentrations as indicated in the results, using the intraperitoneal (i.p.) route. Peritoneal exudate cells (PEC) obtained by peritoneal washings were used as effector cells in the cytotoxicity assay. In some experiments, mice were inoculated with non-virus-containing material derived from non-infected BHK cultures. PEC from these mice did not show higher activity in the cytotoxicity assays than cells from normal mice. Respective data were therefore omitted from the results.

Cytotoxicity assays The slCr-release method (29) with modifications detailed in ref. 30 was used for measuring HSV 2-induced cytotoxic activities. To obtain optimal sensitivity of the assay system, target­ effector-cell mixtures were incubated for about four hours at 37"C, then overnight at 30°C in an atmosphere of 5 % CO 2 in air. Results are expressed as percent specific slCr-release calculated from the geometric means of the counts per minute (cpm) of four replicas using the formula : [(E-LC) : (HC-LC)] X 100. E is the total SICr released by target cells in the presence of the respective effector cells. LC is the low control determined by the activity of normal effector cells which is usually identical or lower than that caused in the presence of culture medium alone, varying between 20 and 40 % . HC represents the amount of isotope-release after incubation of the target cells for 15 minutes at 37°C with 10 % Triton X-IOO (Serva Feinbiochemica, Heidelberg, W. Germany). Standard error of the mean was usually less than 1.05. Experiments have been performed three times or more yielding similar results.

Estimation of the HSV2-LDso Groups of ten male mice (8-10 weeks) were injected i.p. with various doses of infectious HSV 2. Mortality was proto coiled over a period of three weeks. LDso was calculated by the method of SPEARMAN-KARBER (outlined in ref. 31).

Results

Intraperitoneal infection of mice with HSV 2 leads to the death of the animals within 6 to 12 days if the virus dose is high enough. The mice die with symptoms of encephalitis. Table 1 lists several mouse strains in the order of their sensitivity to the lethal effects of an HSV 2 infection. A rough subdivision can be made into sensitive, medium resistant and resistant mouse strains. SWRlJ is according to our experience the most susceptible mouse line followed by AKR/J. C3H/HeJ is the most resistant strain. Table 1 also gives the major histocompatibility types (H-2). The lack of correlation between a certain H-2 allele and natural resistance to HSV2 is at least obvious for H-2k. We have selected ten mouse strains from Table 1 and tested their capability for generating non-antigen-specific cytotoxic cells after i. p.

372 . D. ARMERDING and H. ROSSITER

inoculation with 105 PFU of HSV 2. Mouse tumor lines were used as targets. Peritoneal exudate cells served as effector population. It will be demonstrated elsewhere that cytotoxic responses induced in this manner and assayed in our system are not due to conventional T killer cells, nor to macrophages or antibody-dependent mechanisms, but to cells fulfilling all criteria of NK cells!) as far as they have been defined by others (22-24). Thus significant NK cell activity was found in all mouse strains tested between 3 to 5 days after HSV 2 infection. Table 2 presents the data of one such experiment. Assayed on day 4, all ten mouse strains exhibit good cytolytic responses to Neuro 2A and L 929 tumor cell targets. Cytotoxic activity of the PEC from three of the mouse strains is quite low when tested against P815 targets: two of those are HSV2 resistant (C57Bl!6], C3HI He]), and one is medium resistant (DBA/2). Thus it is not possible to correlate low (or high) NK activity at a late stage after HSV 2 infection with resistance or sensitivity to the same virus. The athymic nude mouse has been reported to exhibit the same resistance to HSV infections as the corresponding wild-type strain (32). It has also been demonstrated that nude mice have high levels of spontaneous NK cells (23, 25). When we tested PEC from normal non-infected nude mice in our system, this normal NK-cell level was as low as in non-mutant mouse Table 1. Sensitivity of various inbred mouse strains to the lethal effects of HSV 2') Mouse strain C )

H-2 type

LDsob) (PFU HSV2)

SWR/J AKR/J

q k

7.9 X lOt 2.0 X 102

DBAI2 Han CBA/J Balblcl A Born A/J DBA/I J C57BRc/Han C3H/Bi Mai C57Bl/Ks J

d k d a q k k k

X 103 X 103

SJLlJ C3H/He/Han C3H/Tif Born C57Bl/I0ScCr/Bom C57Bl/I0 J C57Bl/6 J C3H/He J

k k b b b k

1.6 2.7 1.0 X 1.5 X 1.6 X 2.0 X 3.2 X 4.0 X 1.3 X 1.0 X 2.5 X 3.2 X

104 104 104 104 104 104

Classification

sensitive

medium resistant

lOS lOS lOS

resistant

lOS

6

"" 10 6 "" 10 6 "" 10

') This table is meant to give a rank order of sensitivity of the mouse strains tested against lethal HSV2 infections. The actual LDso might vary for a certain mouse strain from experiment to experiment especially if different virus preparations are used. b) Virus was injected i. p. in 0.2 ml of medium. Mortality was estimated on day 21. C) Only male mice were used. Age was 8-10 weeks. 10 mice per mouse strain were tested per virus dose in this experiment.

NK Cells in HSV-resistant and -sensitive Mice' 373 Table 2. NK-cell activity of various inbred mouse strains on day 4 after HSV2 infection') Percent specific 51Cr release Experiment

mouse strain Neuro 2A 50 : 1 10 : 1

50: 1

10 : 1

50: 1

10 : 1

SWR/] AKR/] DBAI2 ] Balb/c/A Born

41.3 67.3 50.6 51.8

9.8 34.8 12.1 20.9

45.0 50.7 33.1 62.2

13.5 23.7 11.1 17.2

44.6 21.0 7.3 31.5

11.3 19.3 0.0 4.9

II

DBA/l ] C3H/HeHan S]L!] C57BI/6 ]

89.2 43.8 65.9 48.7

26.3 16.4 19.7 28.5

84.8 30.9 64.4 18.5

24.2 18.7 16.3 9.9

15.9 13.1 24.2 7.9

1.1 9.7 12.2

III

C57Bi!10 J C3H/HeJ

65.5 19.8

13.7 9.5

26.5 22.5

29.4 2.6

9.3 7.1

0.0 0.0

L929

P815

3.4

a) PEC were isolated 4 days after i. p. injection of 105 PFU of HSV2.

strains (data not shown). We thus compared the capability to generate NK cells in nude and wild-type mouse strains infected with HSV 2. C57B1I10, CBA, and Balb/c athymic and normal mice were injected i.p. with HSV 2. PEC were tested again on day 4 after infection. Cytotoxicity was assayed against L929 and Neuro 2A-tumor target cells. The results of one typical experiment are documented in Table 3. Note: 1. Cytotoxic responses of nude CBA and Balb/c mice are as high as those of the nonmutant strains. 2. PEC from nude C57B1I10 mice exhibit even higher levels of NK activity than the respective wild-type line. We could demonstrate elsewhere that NK activity in some mouse strains is already high 24 hours after HSV infection!). Since we believe that the Table 3. Induction of NK-cell activity with HSV2 in athymic nude mouse strains') Percent specific SICr release Experi­ ment

Effector cells a ) L929 50: 1

II

III

Neuro 2A 25 : 1

50 : 1

25 : 1

C57Bi!10ScSn C57Bi!10ScSn nu/nu

26.6 100.0

12.4 50.2

27.6 79.8

14.4 14.4

C57Bi!10ScSn C57Bi!10ScSn nu/nu

12.9 23.5

< 1.0 5.1

29.3 22.4

10.6 7.6

CBA/J CBA/Ca nu/nu

53.4 55.3

25.6 16.1

25.9 22.7

19.8 14.8

Balb/c Balb/c nu/nu

13.3 12.9

7.0 11.0

43.5 15.7

11.3 7.3

') PEC were isolated 4 days after i. p. injection of 105 PFU of HSV2.

30

~

tTl

~~ro~~~oe~~~oo~rooo~~

Figure 1. Comparison of different inbred mouse strains with regard to early induction of non-virus specific killer cells after Mice were infected i.p. with 105 PFU of HSV2 24 hours before isolation of PEC.

C3H/HE J

C575 BL/1OJ

C57BL/6J

SJL/J

C3H/HE HAN

DBA/IJ

BALB/C/A 80M

DBA/2J

~ '" ~

;:r:

0-

()

~

L929

>­ :;::

:>'

AKR/J

NEUR02A

P815

>='

Z

TARGET CELLS: 3T6

25:1

.j>.

-..J

W

SWR/J

EFFECTOR CELLS

_

0

EFFECTOR:----------------------------------------------------------------------------------------~ TARGET RATIO 100 : 1

30.9 100.0

100.0 100.0

4

35.7 67.3

53.5 89.0

5

11 .3 34.5

21.9 54.5

0.0 42.0

45.4 35.6

19.5 72.8

4.0 47.8

22.1 26.5

8.2 51.1

0.0 0.0 30.5

0.0 65.4

< 1.0

100.0

50 : 1

100 : 1

50 : 1

100 : 1

25: 1

Neuro 2A

19.1 22.1

0.0 41.3

15.8

25 : 1

0.0 57.8

14.0 100.0 61.0 52.9

25.6

100.0 85.8

< 1.0

50 : 1

L929

0.0 48.9

100 : 1

Percent specific StCr release

3T6

2

Day of assay

') PEC were isolated after i. p . injection of 10 PFU of HSV2 .

SWR/J Balb/c

Balb/c

SWR/ J

SWR/J Balb/c

Mouse strain

Table 4. NK cell responses in SWR/J and Balb/c mice at various times after HSV2 infection')

42.4 37.7

0.0 26.8

0.0 10.7

25 : 1

45.1 80.7

15.3 6 93 .3

0.0 20.4

100 : 1

27.7 39.7

.8 43.6

16.8

< 1.0

50 : 1

P815

8.4 28.3

7.5 35.5

9.7

< 1.0

25 : 1

?"1

VI

-..I

W

'"

r; .

'"~

~.

'"~.

~

0

0-

g

.,P

.,~.

..,~

(Jl

::x:

5·

'":;;:

()

Z

376 . D.

ARMERDING

and H.

ROSSITER

cellular events which take place within this time period are the most important ones for the natural defence, we compared the same ten mouse strains listed in Table 2 for early cytotoxic responses. The results are illustrated in Figure 1. The following findings are noteworthy: 1. Both HSV-sensitive mouse strains tested (SWR, AKR) generate low or no NK­ cell responses. 2. Two of the five resistant mouse lines are also low NK producers. 3. All the other mouse strains are intermediate to highly effective with regard to NK activity. Table 4 reiterates the findings demonstrated above for the HSV2sensitive SWR and the medium resistant Balb/c mice: Both mouse strains exhibit equally high NK-cell activity on day 4 after infection. However, SWR PEC fail to react 24 hours after virus inoculation, whereas Balb/c cells demonstrate good cytotoxic responses. Moreover, tested on day 2 after HSV2 infection, Balb/c PEC exhibit peak responses, whereas SWR/J mice just start to generate NK activity.

Discussion Natural resistance of mice to the lethal effects of an HSV infection has been associated with several non-antigen specific cellular activities. The basis of such studies was the remarkable difference between certain mouse strains with regard to survival of herpes-virus infections. This phenomenon was first described by C. LOPEZ (5) for HSV 1. The data which are presented here for HSV 2, which are similar to those obtained by H. KIRCHNER and coworkers (21, 32), basically correlate with those of LOPEZ. The rank order of sensitivity to HSV 1 and HSV 2 of the various mouse strains tested appears to be the same. Extensive in vitro studies have been carried out in order to ascertain cellular parameters which could be associated with the in vivo natural resistance to HSV or the lack of it. H. KIRCHNER, R. ZAWATZKY, and colleagues (21, 33) suggested that capability for production of interferon determines natural resistance against herpes-virus infections in mice. The authors demonstrated that only resistant mouse strains produce interferon (type 2) in response to HSV 2 in cell culture. Failure of macrophages from HSV -resistant mouse strains to replicate HSV in vitro was considered by several investigators to correlate with the in vivo situation and to be responsible for natural resistance (5, 12, 14, 15, 32). LOPEZ, however, demonstrated that the in vitro and in vivo non-permissiveness of phago­ cytes to herpes replication does not necessarily correlate (14, 34). Hence this shows that it is quite important to study cellular activities induced in vivo rather than those obtained in vitro. The present investigation, there­ fore, deals with the in vivo induction of another potential resistance factor: the generation of NK -cell responses after HSV infections in susceptible and resistant mouse strains.

NK Cells in HSV-resistant and -sensitive Mice· 377

We could show here that with regard to NK -cell activity no marked differences can be found among all the mouse strains tested if assayed on day 4 after HSV 2 inoculation. Also nude mice which have been demon­ strated by ZAWATZKY and coworkers (33) to be as resistant to HSV infections as the corresponding wild-type strains can be effectively stimu­ lated to produce NK activity. However, we believe that the key to understanding the mechanisms of antiviral natural resistance lies in the early events after infection. We have documented here that all-or-none type of responses can be observed within one day after onset of infection. Two mouse strains which are highly susceptible to HSV 2 infections (AKR, SWR) do not generate NK cells in the critical phase of the first 24 hours after virus infection. It is of importance to note that two of the resistant mouse lines, the SJL and the C57Bl/10 mice, do not exhibit NK activity within 24 hours either. One has to assume in the latter case that in these mouse strains other defence mechanisms than NK cells are respons­ ible for resistance. However, lack of early induction of NK cells in these two mouse strains does not prove that these cells do not play an important role in natural resistance in other HSV-non-susceptible lines. In order to study the exclusive role of NK cells in antiviral resistance, two possibilities are promising. First, it should be possible to transfer resistance from one mouse strain to another, analogous to the bone marrow transfer of LOPEZ (25). One would have to preselect for NK cells or NK-cell precursors. Secondly, screening a wide variety of different mouse strains for several in vivo resistance factors such as permissiveness of the host cells for virus-replication, anti-viral macrophage activities, interferon production, and others, it might be possible to find mouse strains lacking one or more of these functions except NK-cell activity. The medium-resistant lines appear to be most interesting in this regard since the highly sensitive strains have additional defects besides their failure to induce early NK-cell responses 2 ). Preliminary results indicate that in fact mouse strains can be found with impaired macrophage functions and reduced ability to produce interferon after virus contact, but which still exhibit high NK activity and are medium resistant to HSV 2 (to be published). Furthermore, a thorough genetical analysis - in continuation of the work of C. LOPEZ (5) - should also give insight into the role and regulation of virus-induced NK cells in anti-viral resistance. Finally, another alternative way to ascertain the role of particular cellular functions in the host defence to viral infections should be mentioned. LOPEZ and coworkers demonstrated recently that by treatment with 89Sr of HSV 1-resistant mice they do not only abrogate resistance, but also a bone­ marrow(M)-dependent cell function which resembles that of NK cells (35). It has also been shown before by O. HALLER and H. WIGZELL that such 2) ARMERDING, D., P. MAYER, M. SCRIBA, and H. ROSSITER. The role of macrophages and natural killer cells in sustaining genetically determined resistance to herpes simplex virus type 2 in inbred mice. Manuscript in preparation.

378· D. ARMERDING and H. ROSSITER

treatment suppresses NK-cell activity (36). LOPEZ et al. concluded from their results that at least in the mouse strain used resistance is mediated by M cells. Further studies are necessary to elucidate the relation between NK and M cells and to compare their function in different mouse strains. Acknowledgements The authors are thankful to Ms. ANDREA HREN for technical assistance, to Dr. HOLGER KIRCHNER for helpful discussion and to Dr. MARIANNE SCRIBA for stimulating criticism.

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