macrophage-dependent focal liver necrosis, which was only demonstrable after intraperitoneal ..... to HSV-2-induced necrotic hepatitis, readily adsorbed HSV-2,.
Vol. 37, No. 3
INFECTION AND IMMUNITY, Sept. 1982, p. 907-911
0019-9567/82/090907-05$02.00/0 Copyright C 1982, American Society for Microbiology
Early Interactions of Herpes Simplex Virus with Mouse Peritoneal Macrophages BO SVENNERHOLM, ANDERS VAHLNE, AND ERIK LYCKE* Department of Virology, Institute of Medical Microbiology, University of Goteborg, Goteborg 413 46, Sweden
Received 21 October 1981/Accepted 27 April 1982
Adsorption of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to resident peritoneal macrophages (PM) of 4-week-old Swiss albino (SA) and GR! AFib mice was studied. A significantly (P < 0.05) higher HSV-2 adsorption rate was found with PM of SA mice than with PM of GR/AFib mice. Of added HSV-2, 65% bound to the cells of SA mice over a 120-min period versus 15% to PM of GR/ AFib mice. Only 15 to 20o of added HSV-1 bound to PM regardless of the mouse strain. These patterns of adsorption were found with all four HSV-1 and four HSV-2 strains tested. Pretreatment of PM with an HSV-2 mutant blocked the adsorption of added HSV-2. Thus, the receptors for HSV attachment seemed to be virus type selective. To avoid masking of adsorption by phagocytotic activity, the adsorption studies had to be performed at 4°C. Transport of attached HSV-1 and HSV-2 to the nuclei of SA PM was studied with purified virus labeled with 32Pi and [3H]thymidine. In double-isotope experiments, only transport of HSV-2 was detected. The possible importance of differences in density or avidity of virus-binding receptors on the plasma membrane of PM is discussed in relation to macrophage-dependent focal liver necrosis, which was only demonstrable after intraperitoneal inoculation of HSV-2, not HSV-1, only in SA, not GR/AFib, mice. It is generally accepted that macrophages are of primary importance for naturally occurring resistance to virus infections (1, 11, 15). Resistance of mice to experimental herpes simplex virus (HSV) infection can be abrogated by depletion of the macrophage population (16, 29), whereas macrophage-stimulating agents increase resistance to the virus (7, 10). However, the mechanisms by which macrophages participate in host defense reactions are not yet fully understood. Macrophages can transfer capacity of resistance to HSV infection to other cells (17, 18). Resident macrophages isolated from adult animals of most mouse strains are not permissive for HSV (6, 15) unless precultivated for 3 to 4 days in vitro (9), but macrophages from young animals can be productively infected with HSV and yield infectious progeny virus (4). Such an age-dependent HSV resistance also exists for human macrophages (12). Despite their genetic relationship, HSV type 1 (HSV-1) and HSV type 2 (HSV-2) infections have striking differences in pathology. For example, when injected intraperitoneally, HSV-2 but not HSV-1 will, in most mouse strains, induce focal necrotic lesions on the surface of the liver (13, 26). This distinguishing characteristic has been reported to be associated with a
restricted HSV-1 replication in peritoneal macrophages (PM) (14), and likewise PM from HSV1-resistant mouse strains restrict HSV-1 replication significantly better than macrophages of susceptible mice (9). The present study elucidates some early virusPM interactions, i.e., the attachment of virus to macrophages and the processing of HSV from the macrophage plasma membrane to the nucleus. The possible importance of HSV-binding receptors on the plasma membrane of PM for observed resistance to HSV-1 and HSV-2 infections is discussed.
907
MATERIALS AND METHODS Viruses. HSV-1 strain F was obtained from B. Roizman. HSV-1 strain KJ502 was isolated in our laboratory (26). Two HSV-2 strains (B4327UR and 7875) were supplied by S. Jeansson. Additional strains of both HSV-1 and HSV-2 were used. The latter strains were newly isolated in our laboratory and had a low passage history (two to three passages). All strains employed were plaque purified and typed by immunoelectroosmophoresis against type-specific antisera (5). A trisodium phosphonoformate (PFA)-resistant strain of HSV-2 was used for receptor blocking experiments
(24, 27). Plaque titrations of infective virus were carried out on monolayer cultures of GMK AH-1 cells grown in 5-
908
plastic dishes with Eagles minimal essential medi(MEM) supplemented with 2% calf serum, 1% methylcellulose, and antibiotics as an overlaying medi-
cm
um
um.
HSV
INFECT. IMMUN.
SVENNERHOLM, VAHLNE, AND LYCKE
was
labeled with 1 mCi of [3H]thymidine
or
32p, (The Radiochemical Center, Amersham, England) and was partially purified. The overlay medium of infected cells, as well as cellular fractions that were hypotonically (1 mM phosphate-buffered saline [pH 7.2]) blended in a Dounce homogenizer, were pooled and clarified by low-speed centrifugation. Subsequently, the supernatant was centrifuged at 15,000 x g for 5 min and chromatographed on a Sepharose C-L 2B column as described previously (23). Fractions corresponding to the void volume containing all infectivity and a peak of radioactivity were collected. Mice. When not otherwise specified, Swiss albino (SA) mice from our own laboratory strain were used. In addition, inbred specific-pathogen-free GR/AFib mice obtained from the Fibiger Laboratory, Copenhagen, Denmark, were used. Isolation of macrophages from all mice was performed when the mice were 4 weeks old. Macrophages. Isolation of PM from unstimulated mice was performed by standard procedures (14). Briefly, unstimulated resident PM were obtained by peritoneal lavage with 3 ml of Eagle MEM per mouse. Cells of peritoneal exudate were added to plastic tissue culture bottles (Nunc, Copenhagen, Denmark) and incubated for 2 h at 37°C, and nonadhering cells were removed by washing the monolayers five times with Eagle MEM. Subsequently, the cells were incubated overnight in Eagle MEM containing 20% fetal calf serum, 200 U of penicillin, and 200 ,ug of streptomycin per ml. As judged by plastic adherence, morphological appearance, and acridine orange fluorescence, 95% of remaining cells were considered macrophages. Adsorption of HSV to macrophages. The kinetics of the attachment of HSV to suspended macrophages was studied by assaying residual nonadsorbed virus infectivity as described perviously (28). The suspensions of cells were obtained by gently scraping the monolayer of macrophages with a rubber policeman. In each experiment, 107 5 x 105 cells were suspended in 0.6 ml of Eagle MEM in sterile plastic tubes pretreated with bovine serum albumin (2%) for 1 h at 37°C to avoid adherence of macrophages to plastic surfaces. Suspensions were agitated at 4 or 37°C. Samples were drawn immediately after the addition of 106 plaque-forming units (PFU) of HSV and then after intervals of 15, 30, 60, and 120 min. Each sample (100 ,ul) was added to 9.9 ml of ice-cold medium and spun at 1,000 x g for 10 min. Residual infectivity in the supernatant was titrated. Residual infectivity of cellfree controls was determined by the same procedure as described above. Penetration and transport of HSV to the cell nucleus. In double-isotope experiments, the processing of attached virus from the cellular plasma membranes to the nuclei of PM was studied by the simultaneous infection of the same population of cells with both HSV-1 (labeled with [3H]thymidine or 32p,) and HSV-2 (labeled with [3H]thymidine or 32p;). Purified virus ([3H]HSV-1 and [32P]HSV-2, or vice versa; 2 x 105 to 5 x 105 cpm and 1 x 106 to5 x 106 PFU of each type of HSV) was added to monolayers of 107 PM. After adsorption at 4°C for 2 h, the cells were washed four ±
TABLE 1. HSV-induced liver necrosis 5 days after infection of SA and GR/AFib mice' No. of mice with necrosis
Mouse strain
(total no. of mice) HSV-1
HSV-2
SA 0 (8) 6 (9) GR/AFib 0 (8) 0 (9) a Four-week-old mice were inoculated intraperitoneally with 105 PFU of HSV.
times and incubated at 37°C. Immediately, and after 4 h of incubation, cells were scraped off with a rubber policeman and suspended in Hanks buffer. A fraction (1/10) was collected for determination of the total cellassociated radioactivity. Nuclei from the remaining suspension of PM were purified by the method of Blobel and Potter (2). The percentage of cell-associated radioactivity found in the nuclei was calculated. RESULTS
Appearance of HSV-induced macroscopic necrotic foci on the mouse liver surface. Four-weekold mice of SA and GRIAFib strains were inoculated intraperitoneally with 105 PFU of HSV-1 (F) or HSV-2 (B4327 UR). Five days after infection, the mice were examined for the purpose of macroscopic necrotic foci on the liver surface. Several distinct necrotic lesions were observed in 67% of the HSV-2-inoculated SA mice (Table 1). However, all HSV-2-inoculated GR/AFib-mice were devoid of macroscopically demonstrable foci. After infection of mice with HSV-1, necrotic foci on the liver surface were not observed. The results are in close agreement with findings reported by Mogensen (15). Adsorption of HSV to macrophages. The attachment of HSV-1 and HSV-2 at 37°C to suspended PM or SA mice is shown in Fig. 1. Determinations of residual nonadsorbed infectious virus revealed a relatively high loss of infectivity due to inactivation of HSV and nonspecific adsorption, as indicated by the cell-free controls. After adjustment for the nonspecific reduction of infectivity, a specific attachment of HSV-2 but not HSV-1 to PM was demonstrable. To overcome the effects of heat inactivation and the adherence of virus to plastic surfaces of the bottles and tubes used and to reduce the uptake of virus by the macrophage phagocytotic activity, attachment studies were performed at 4°C with a prolonged incubation time. In the cell-free controls, an average of 5 x 104 PFU (5%) disappeared in 120 min at this temperature. Figure 2 shows adsorption profiles of four HSV1 and four HSV-2 strains to PM of HSV-2susceptible (SA) and -resistant (GR/AFib) mice. The attachment of HSV to both populations of PM had a distinct and reproducible pattern. The adsorption rate was essentially the same for the
VOL. 37, 1982
HSV EARLY INTERACTIONS WITH MACROPHAGES
le~~~~~~~~
2
00
>
o
(/
minutes FIG. 1. Adsorption of HSV-1 (O, *) and HSV-2 (E, A) to suspended macrophages of SA mice (--- ) at 37°C. (-) Corresponding cell-free controls of HSV-1 and HSV-2. L._
four strains of each HSV type. There were, 0~~~~type-related differences. The PM cells however, of SA mice adsorbed 65% of added HSV-2 after 120 min, whereas only 15% of HSV-1 bound to
100-
tn
A=
15 30
60 minutes
120
FIG. 2. Adsorption profiles of four HSV-1 strains (A) and four HSV-2 strains (A) to suspended PM of HSV-susceptible (SA; solid lines) and -resistant (GR/ 4oC. mice at Plotted curves were AFib; dashed lines) adjusted with regard to results of cell-free controls. Bars, Standard errors of the mean.
909
the cells. In contrast, only 15 to 20% of HSV-1, as well as of HSV-2, adsorbed to PM of GR/ AFib mice. Thus, a significantly higher (P < 0.05) adsorption rate of HSV-2 was encountered with PM of susceptible SA mice as compared with resistant GR/AFib mice. A low HSV-1 adsorption rate was found with PM of both mouse strains. Analogous results were obtained when radiolabeled viruses attached to PM were used as a parameter (data not shown). Control experiments were performed to determine whether that attachment rates could be influenced by differences between the two virus types in sensitivity to proteolytic enzymes possibly released to the suspending mediumn from macrophages. Lysates of SA macrophages from which the membranes or contaminating cells had been removed were incubated with HSV for 2 h at 4 and 37°C, respectively. In tests of two laboratory strains of each type, the loss of infectivity was in no case larger than that of the cell-free controls. The result is in agreement with the findings of Morahan et al. (18). HSV type-selective receptors on macrophages. The adjusted titers of residual virus infectivity of adsorption experiments with HSV-2 to PM at 4°C were considered to reflect attachment of virus to virus-binding receptors on the plasma membrane of macrophages. This assumption was based on the following type of experiment: PM of HSV-2-susceptible SA mice were treated five times with drug-sensitive (PFA) type 2 virus at a high multiplicity of infection (20 PFU/cell) in a 3-h period at 4°C. The attachment rate of PFAresistant HSV-2 mutant was then tested, plaquing nonadsorbed virus in the presence of 0.5 mM PFA. At this concentration of the antiviral drug in the overlaying medium, the plaque production of the PFA-sensitive virus was reduced by 4 log units. Pretreatment of cells with PFA-sensitive type 2 virus significantly inhibited the adsorption of the added PFA-resistant virus (Fig. 3). The attachment rate was compared with that of PFA-resistant virus with untreated cells. The findings were consistent with the previously reported blocking of HSV attachment by pretreatment of other cell lines with homotypic virus (27). In contrast, pretreatment of PM with HSV-2 did not influence HSV-1 adsorption to the cells (data not shown). Penetration and transport of HSV to the cell nucleus. In three double-isotope experiments, the processing of cell-associated HSV-1 and HSV-2 to the cell nucleus of SA PM was studied (Table 2). HSV-1 and HSV-2 were labeled with 13H]thymidine and 32p;, respectively, or vice versa. The percentage of total radioactivity found in the nuclear preparations after 4 h at 37°C indicated that HSV-2 was processed much more efficiently than HSV-1. In addition, the
910
SVENNERHOLM, VAHLNE, AND LYCKE
(13). This biological marker, distinguishing the two types of HSV, has been shown to reflect permissiveness of PM for HSV-2 but not HSV-1 (14). Inbred mouse strains have been claimed to exhibit a genetically controlled susceptibility to this HSV-2-induced, macrophage-dependent necrotic hepatitis (13, 15). In contrast, the view of a genetically controlled macrophage resistance has been challenged by Lopez and Dudas (8, 9), who found that although PM of the resistant mouse strain (C57B1/6) restricted HSV-1 better than PM of the susceptible A/g mice, PM of the resistant (C57 x A/g) Fl mice did not restrict HSV-1 replication. In the present report, we have demonstrated that SA mice, susceptible to HSV-2-induced necrotic hepatitis, readily adsorbed HSV-2, whereas GR/AFib mice, which are resistant to the HSV-induced hepatitis, adsorbed significantly less infective HSV-2. In addition, this decreased absorption occurred at a markedly
50-~~~~ C_
15 30
120 lower rate. Although more extensive studies are
60
minutes FIG. 3. Adsorption of the HSV-2 PFA-resistant mutant to suspended PM of SA mice pretreated with PFA-sensitive HSV-2 (0) and to untreated macrophages (O). Plaque assays were performed in cultures with fluid medium containing 0.50 mM PFA.
relative increase of radiolabeled type 2 virus DNA in the nuclear fractions during the first 4 h postadsorption was three to four times larger than the amounts demonstrable with HSV-1 (P < 0.005; Student's t test). DISCUSSION Strains of HSV-2 but not HSV-1 produce a focal, macroscopically demonstrable necrotic hepatitis in intraperitoneally inoculated mice TABLE 2. Percentage of cell-associated radioactive HSV DNA of nuclear preparations of PM' Expt
% HSV DNA
Relative increase in
radioactive at 4 h
radioactivityb(Oto4h)
HSV-1
INFECT. IMMUN.
HSV-2
HSV-1
ic
HSV-2
12.3 85.2 0.8 3.7 2.9 25.2 0.8 2.8 3c 9.3 56.5 1.1 2.9 a In double-isotope experiments, penetration to the nuclei 4 h postadsorption was studied after simultaneous infection of PM with radiolabeled HSV-1 and HSV-2. b Counts per minute at 4 h/counts per minute at 0 h. c [3H]thymidine-labeled HSV-1; HSV2. d 32P,-labeled HSV-1; [3H]thymidine-labeled HSV2.
2d
32Pi-labeled
needed before the importance of virus-binding macrophage receptors can be evaluated, the observation suggests that the presence or absence of virus-binding receptors on the plasma membrane may be one discriminating factor that influences the susceptibility or resistance of macrophages to HSV infection. Cellular receptors for HSV are virus type selective (27). Therefore, the blocking of the adsorption to PM of one HSV-2 strain by means of another type 2 strain would emphasize that the virus was adsorbed to specific receptors. However, to avoid interference of phagocytosis, the experiments were performed at 4°C, at which temperature essentially all the phagocytotic activity is abolished. Previously, Stevens and Cook (22) have reported that PM of 8-weekold BRVS Swiss-Webster mice adsorbed HSV-1 less efficiently than permissive RK 13 cells. The macrophages of the mouse strain observed by these authors became abortively infected with HSV-1 but synthesized viral DNA and proteins. The restriction of HSV infection was therefore suggested to be at the level of virus assembly. In addition to Stevens and Cook, a number of other authors have considered whether the lack of virus-binding receptors could be a mechanism for resistance to infection (4, 12, 14, 21, 25) or whether an enhanced virus adsorption by activated macrophages could act as an antiviral effect of PM (19). However, in none of the studies has a difference in adsorbing capacity between resistant and susceptible PM been reported. Adsorption studies with PM offer other problems in addition to those of virus inactivation and nonspecific adherence. To avoid masking of the adsorption by macrophage phagocytosis, the
VOL. 37, 1982
HSV EARLY INTERACTIONS WITH MACROPHAGES
experiments must be performed at a temperature at which the phagocytotic activity is arrested. Peptone-stimulated PM of adult mice, for example, are markedly more active in both uptake
911
against herpes simplex virus infection in mice by Corynebacterium parvum. Infect. Immun. 16:9-11. 8. Lopez, C. 1975. Genetics of natural resistance to herpesvirus infections in mice. Nature (London) 258:152-153. 9. Lopez, C., and G. Dudas. 1979. Replication of herpes simplex virus type 1 in macrophages from resistant and and destruction of HSV (4) than are unstimulatmice. Infect. Immun. 23:432-437. ed macrophages. Moreover, it is probably nec- 10. susceptible Martinez, D., R. J. Lynch, J. B. Meeker, and A. K. Field. essary to use the adherent peritoneal cell popu1980. Macrophage dependence of polyriboinosinic acidlation only, to reduce the influence of cell polyribocytidylic acid-induced resistance to herpes simplex virus infection in mice. Infect. Immun. 28:147-153. heterogeneity, even though this population 11. Mims, C. A. 1964. Aspects of the pathogenesis of virus might not be completely homogeneous (18). diseases. Bacteriol. Rev. After virus adsorption, transport of attached 12. Mintz, L., W. L. Drew, R.28:30-71. Hoo, and T. N. Finley. 1980. virus to the nucleus of the infected cell is reAge-dependent resistance of human alveolar macrophages to herpes simplex virus. Infect. Immun. 28:417quired for a productive infection. Although some HSV-1 bound to the PM, we were not able 13. 420. Mogensen, S. C. 1974. Focal necrotic hepatitis in mice as to detect any transport of virus to the nuclei of a biological marker for differentiation of herpes virus receptor to low in addition these cells. Thus, hominis type 1 and type 2. J. Gen. Virol. 25:151-155. density and avidity for HSV-1 on PM, the proc- 14. Mogensen, S. C. 1977. Role of macrophages in hepatitis induced by herpes simplex virus types 1 and 2 in mice. essing of attached virus seemed to be impaired. Infect. Immun. However, in contrast to the findings with HSV- 15. Mogensen, S. C.15:686-691. 1979. Role of macrophages in natural 1, these two early virus-host cell interactions resistance to virus infections. Microbiol. Rev. 43:1-26. were unimpaired in HSV-2-infected cells. It has 16. Mogensen, S. C., and H. K. Andersen. 1977. Effect of silica on the pathogenic distinction between herpes simbeen suggested that for the productive infection plex virus type 1 and 2 hepatitis in mice. Infect. Immun. of HSV, the virus penetrates the infected cell by 17:274-277. fusion of the virus envelope with the plasma 17. Morahan, P. S., L. A. Glasgow, J. L. Crane, and E. R. Kern. 1977. Comparison of antiviral and antitumor activimembrane (3, 20). Perhaps, due to a low avidity of activated macrophages. Cell. Immunol. 18:404-415. or low abundance of HSV-1 receptors on PM, 18. tyMorahan, P. S., S. S. Morse, and M. B. McGeorge. 1980. the contact between the HSV-1 envelope and Macrophage extrinsic antiviral activity during herpes simplex virus infection. J. Gen. Virol. 46:291-300. the plasma membrane is not close enough to allow fusion to take place. The possibility that 19. Morse, S. S., and P. S. Morahan. 1981. Activated macrophages mediate interferon-independent inhibition of herabortion of HSV-1 infection of macrophages is pes simplex virus. Cell. Immunol. 58:72-84. the result of virus uptake via phagocytosis and a 20. Sarmiento, M., M. Haffey, and P. G. Spear. 1979. Membrane proteins specified by herpes simplex viruses. III. subsequent lysosomic degradation of the virus Role of glycoprotein VP7(B2) in virion infectivity. J. Virol. will be the subject of further investigations. 29:1149-1158. 21. Shif, I., and F. B. Bang. 1970. In vitro interaction of mouse hepatitis virus and macrophages from genetically ACKNOWLEDGMENTS resistant mice. I. Adsorption of virus and growth curves. We are indebted to A.-S. Tylo for skillful technical assistJ. Exp. Med. 131:851-862. ance. 22. Stevens, J. G., and M. L. Cook. 1971. Restriction of This research was supported by the Swedish Medical Reherpes simplex virus by macrophages. An analysis of the search Council (grant no. 4514) and by the Medical Faculty of cell-virus interaction. J. Exp. Med. 133:19-38. the University of Goteborg. 23. Svennerholm, B., A. Vahine, S. Jeansson, R. Lunden, S. Olofson, G. Svantesson, and E. Lycke. 1980. Separation of herpes simplex virions and nucleocapsids on Percoll graLITERATURE CITED dients. J. Virol. Methods 1:303-309. 1. Allison, A. C. 1974. On the role of mononuclear phago- 24. Svennerholm, B., A. Vahine, and E. Lycke. 1979. Inhibitory effect of trisodium phosphonoformate on HSV in cytes in immunity against viruses. Prog. Med. Virol. tissue cultures. Proc. Soc. Exp. Biol. Med. 61:115-118. 18:15-31. 2. Blobel, G., and R. van Potter. 1966. Nuclei from rat liver; 25. Turner, G. S., and R. Ballard. 1976. Interaction of mouse peritoneal macrophages with fixed rabies virus in vivo and isolation method that combines purity with high yield. in vitro. J. Gen. Virol. 30:223-231. Science 154:1662-1665. 3. DeLuca, N., D. Bzic, S. Person, and W. Snipes. 1981. 26. Vahlne, A., J. Blomberg, S. Olofsson, and E. Lycke. 1975. Subtyping of herpes simplex virus. Acta Pathol. MicrobiEarly events in herpes simplex virus type 1 infection: ol. Scand. Sect. B 83:506-512. photosensitivity of fluorescein isothiocyanate treated viri27. Vahlne, A., B. Svennerhohn, and E. Lycke. 1979. Evions. Proc. Natl. Acad. Sci. U.S.A. 78:912-916. dence for herpes simplex virus type selective receptors on 4. HIch, M. S., B. Zsmnan, and A. C. Allison. 1970. Macrocellular plasma membranes. J. Gen. Virol. 44:217-225. phages and age-dependent resistance to herpes simplex 28. Vahlne, A., B. Svennerholm, M. Sandberg, A. Hamberger, virus in mice. J. Immunol. 104:1160-1165. and E. Lycke. 1980. Differences in attachment between 5. Jeansson,S. 1972. Differentiation between herpes simplex herpes simplex type 1 and type 2 viruses to neurons and virus type 1 and type 2 strains by immunoelectroosmoglial cells. Infect. Immun. 28:675-680. phoresis. Appl. Microbiol. 24:96-100. 6. Johnson, R. T. 1964. The pathogenesis of herpes virus 29. Zlsman, B., M. S. HIrsch, and A. C. Allison. 1970. Selective effects of antimacrophage serum, silica and antilymencephalitis. II. A cellular basis for the development of phocyte serum on the pathogenesis of herpesvirus infecresistance with age. J. Exp. Med. 120:359-374. tion in young adult mice. J. Immunol. 104:1155-1159. 7. Kirchner, H., H. M.Hirt, and K. Munk. 1977. Protection
