MACROPHAGE ACTIVATION IN MICE INFECTED WITH ECTROMELIA OR LYMPHOCYTIC CHORIOMENINGITIS VIRUSES

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1 AJEBAK 51 (Pt. 3) (1973) MACROPHAGE ACTIVATION IN MICE INFECTED WITH ECTROMELIA OR LYMPHOCYTIC CHORIOMENINGITIS VIRUSES by R. V. BLANDEN AND C. A. MIMS' (From the Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T ) (Accepted for publication October 24, 1972.) Summary. Macrophage activation, as demonstrated by an increased ability to kill a bacterium {Listeria monocytogenes) occurred in vims-infected mice. The phenomenon was present in the liver, spleen and peritoneum eight days after infection with lymphocytic choriomenlngitis virus and in the liver and peritoneum eight days after ectromelia virus infection. The mechanisms of macrophage activation, its relationship to the cell-mediated immune response and its possible significance in viral infection are discussed. INTRODUCTION. Macrophages play important roles in host defence against a wide variety of infectious agents including bacteria (Mackaness and Blanden, 1967) and viruses (Mims, 1964). During the course of some infections macrophages become hyperactive or activated, in that their capacity to inactivate ingested organisms is increased (Mackaness and Blanden, 1967). This enhanced activity has been demonstrated to be at least partly non-specific; thus, activated macrophages wliich arise during infection with one agent are effective against other agents (Blanden, 1968). However, in many examples of cross-protection reciprocity has not been investigated, and, in particular, viral infections have not yet been shown to induce macrophage activation. This report presents evidence that maerophage activation, as indicated by an increased ability to kill a bacterium, Listeria monocytogenes, occurs during infection with both ectromelia virus and lymphocytic choriomeningitis (LCM) virus. MATERIALS AND METHODS. Mice. CBA mice obtained from a breeding colony in this laboratory were used in all experiments as young adults. In individual experiments all mice were of the same age and sex. ' Present address: Guy's Hospital Medical School, London Bridge, London, S.E.I. England.

2 394 R. V. BLANDEN AND C. A. MIMS Virus strains. The attenuated Hampstead egg strain of ectromelia virus and the WEg strain of LGM virus were used. Virus stocks were supernatants of saline suspensions of infected chorioallantoic membranes from 14-day-old chicken embryos (ectromelia virus), or infected lungs obtained from guinea-pigs 6-7 days after subcutaneous infection (LGM virus). All stocks were stored in small portions at 70. Ectromelia virus was titrated using a plaque assay on L cells (Blanden, 1970) and amounts of virus expressed in plaque-forming units (PFU). LGM virus was titrated by intracerebral inoculation of mice of the Walter and Eliza Hall Institute strain and amounts of virus expressed as intracerebral LDgQ units. Bacteria. The strain of Listeria monocytogenes used was obtained from Dr. V. P. Ackerman. It was virulent for GBA mice (intravenous LDgQ approx. 2 x 10< viable bacteria). A stock culture was divided into small portions and stored at 70 ; each time a bacterial inoculum for injection into mice was required, a portion was thawed and used to seed a fresh culture. The preparation of bacterial inocula and the enumeration of viable bacteria in tissue, blood and cejl samples from mice has been described in detail previously (Blanden, Lefford and Mackaness, 1969). Evaluation of peritoneal macropliage activity. Groups of 4 or 5 mice were injected intraperitoneally with 6-2 x lo^ viable Listeria per mouse in a volume of 0-1 ml Eagle's minimal essential medium (MEM) with 1% foetal calf serum (EGS). After 7-8 min the mice were killed and their peritoneal cavities washed out with 2 ml of ice-cold MEM with 1% EGS and 10 international units of heparin/ml. Equal volumes of recovered washings (containing free bacteria, maerophages with or without adherent and ingested bacteria and non-phagocytic cells) from individual mice of a group were pooled, and a sample of each pool was diluted 1/5 in MEM with 1% EGS at 4. The remainder of each pool was incubated at 37 for 30 min after which a second sample was diluted 1/5 into MEM with 1% EGS at 4. Both the first (0 min) and second (30 min) diluted samples were then processed as described below to determine the numbers of cellassociated bacteria. The samples were centrifuged (420 g) at 4 for 5 min to deposit cells but not free bacteria (Blanden, Mackaness and Gollins, 1966). Duplicate 0-1 ml samples of a 1/10 dilution of the supernatants were plated on agar to determine numbers of free bacteria. The cell pellet was then disrupted and the cell-associated bacteria resuspended by 10 sec treatment with ultrasound (Measuring and Scientific Equipment Ltd., London, England). Duplicate 0-1 ml samples of a 1/10 dilution of this suspension were plated to obtain a total bacterial count. Subtraction of the numbers of free bacteria from the total counts gave the numbers of cell-associated bacteria at 0 min and 30 min; these numbers indicated the relative abilities of the different peritoneal macrophage populations to inactivate bacteria. The 30 min interval was selected to minimize bacterial multiplication, while allowing time for the cells to kill bacteria. Microscopic examination of the peritoneal cell populations from virus-infected mice showed no increase in the number of polymorphonuclear phagocytes. RESULTS. The effects of viral infection on the antibacterial activity of macrophages in the liver and spleen. After intravenous injection Listeria monocytogenes is rapidly cleared from the blood of normal mice by the littoral macrophages of the liver and, to a lesser extent, the spleen. The antibacterial activity of these macrophages and others which subsequently enter the developing lesions can therefore be evaluated by following the changes in viable bacterial counts in the liver and spleen after intravenous injection (Blanden et al, 1969).

3 MACROPHAGE ACTIVATION IN VIRAL INFECTION 395 Groups of CBA mice were infected intravenously with either 1 x 10^ PFU of ectromelia virus or 1 x 10* LDgo of LCM virus. s remained uninfected. Eight days later all mice were injected intravenously with 9-7 x 10* viable Listeria. After 5 min 5 mice of each group were bled from the retro-orbital plexus, and at 7 min their livers and spleens were removed for determination of viable counts of bacteria in the blood and organs. Further subgroups of 5 mice were sacrificed for liver and spleen counts 4 h and 24 h after injection. Numbers of Listeria in the blood 5 min after injection were similar in all groups (Table 1). Liver counts at 7 min were similar in control and ectromeliainfected mice, but significantly lower (P < as determined in * test) in mice. Subsequent experiments revealed that in mice the lungs contained a much larger portion of the bacterial inoculum than normal mice, thus accounting for this discrepancy. Spleen counts at 7 min were significantly lower than controls in both ectromelia-infected (P <0-01) and (P < 0-05) mice. These differences in the numbers of bacteria initially implanted in the organs had to be taken into account in subsequent evaluation of the behaviour of Listeria over the following 24 h. Comparisons between groups were therefore made using an analysis of variance test of the significance of the differences in the changes in bacterial counts over time intervals. This test revealed that the decline in bacterial numbers in the liver from 7 min to 4 h was significantly greater (P < 0-001) in ectromelia-infected mice than in either of the other two groups; the minor changes in the spleen counts over this interval were statistically insignificant. Growth of bacteria occurred in both the livers and spleens of all mice over the interval from 4 h to 24 h. In the livers this growth was significantly less in ectromelia-infected TABLE 1. The effects of infection of mice with ectromelia or LCM viruses* on the distribution and behaviour of intravenously injected Listeria monocytogenesf. Time after Listeria injection 5 min 7 min 4h 24 h Mice Blood Viable countsf oilisteria in: Liver ± ± Spleen ±O-O ± * 1 X 10' PFU ectromelia virus or 1 x 10^ LDjo LCM virus given intravenously 8 days before Listeria injection. t 9-7 X 10* viable Z.w?m"a. % Mean log viable bacteria per organ + standard error of the mean in groups of 5 mice.

4 396 R. V. BLANDEN AND C. A. MIMS mice than in the other two groups (P < 0-001); growth in livers of LCMinfected mice was also significantly lower than in controls (P < 0-01). On the other hand, the spleens of mice retarded Listeria growth in the interval from 4 h to 24 h significantly more than the other two groups (P < 0-001); ectromelia-infected mice were not statistically different from controls in this respect. Other experiments of similar basic design were performed on mice 5 and 6 days after ectromelia infection, but no significant effect on the behaviour of Listeria in either liver or spleen was found at these times. The foregoing results suggested that the macrophages which engaged Listeria in the livers of mice infected with either ectromelia or LCM virus were activated, but in the spleen this effect was evident only during LCM infection. During infection with other agents the free macrophages of the peritoneal cavity also become activated (Mackaness and Blanden, 1967). Therefore this phenomenon was investigated in the present studies. The effects of viral infection on the antibacterial activity of peritoneal macrophages. Croups of CBA mice were infected intravenously with either 1 x 10^ PFU of ectromelia virus or 1 x 10^ LD50 of LCM virus, or were left uninfected. Eight days later all mice were injected intraperitoneally with Listeria to test the bactericidal capacity of their free peritoneal macrophages, as described in "Materials and Methods". The numbers of viable Listeria associated with the peritoneal macrophages of normal mice did not change substantially over a 30 min interval in vitro (Table 2). The macrophages from both and ectromelia-infected mice killed a significant proportion of cell-associated Listeria over this interval when compared with normal macrophages in a chi-squared test (P < and P < 0-02 respectively). The difference between and ectromelia-infected mice was not significant at the 5% level in this experiment (0-10 > P > 0-05). It is worthy of mention that the peritoneal washing procedure used here did not recover all of the viable bacteria which were injected. More than 50% TABLE 2. The effect of infection* of mice with ectromelia or LCM viruses on the bactericidal activity of their peritoneal macrophages. Mice Numbers of cell-associated viable bacteriaf Omin min *1 X 10'PFU ectromelia virus or 1 x 10'LD50 LCM virus given intravenously 8 days before testing peritoneal macrophages. t Derived by subtracting numbers of free bacteria from total bacteria before (0 min) and after (30 min) incubation of infected peritoneal macrophages in vitro at 37 for 30 min, as described in "Materials and Methods".

5 MACROPHAGE ACTIVATION IN VIRAL INFECTION 397 remained in the animal, perhaps within cells which were not washed out, or were inactivated rapidly before washing was completed. The total recovery from virus-infected mice was less than from normal mice, and this is reflected in the lower cell-associated counts at 0 min (Table 2). However, this fact does not invalidate the data obtained with the populations of macrophages and bacteria which were recovered in the peritoneal washings. DISCUSSION. The evidence presented here shows that macrophage activation occurs during viral infection, but gives little indication of when it arises or how long it persists. In ectromelia-infected mice it was not detectable at 5 and 6 days after intravenous infection, but was present by day 8. mice were investigated only on day 8. For reasons discussed below, it seems likely that the temporal sequence of macrophage activation is linked to the development of the cell-mediated immune response which occurs during both ectromeiia (Blanden, 1970; 1971a) and LCM (Mims and Blanden, unpublished data) infections and is responsible for a potent antiviral effect. The events which lead to macrophage activation during infection have not been fully defined, but recent evidence in vitro suggests that the sensitized lymphocytes which effect cell-mediated immunity (CMI) can, upon specific antigenic stimulation, improve the ability of macrophages to kill bacteria (Simon and Sheagren, 1971), to inhibit bacterial growth (Patterson and Youmans, 1970; Godal, Rees and Lamvik, 1971), to phagocytose bacteria or starch granules (Nathan, Karnovsky and David, 1971), to spread and move on a plastic surface (Mooney and Waksman, 1970; Nathan et al, 1971) and to engage in certain metabolic processes (Nathan et al, 1971). Whether activated macrophages are completely non-specific or possess a degree of specificity in their anti-microbial effects also requires more investigation. In vivo, macrophage activation could well occur most efficiently within foci of infection where essential components of the activating mechanism, sensitized lymphocytes, antigen, macrophages and perhaps other cell types are in close contact (Blanden, 1971b). Since the phenomenon may involve macrophages such as those in the peritoneal cavity which are remote from the sites of infection, a soluble blood-borne mediator of CMI may be involved. There is evidence that soluble mediators of CMI have various effects upon macrophage behaviour (Nathan et al, 1971; Godal et al, 1971), but more work in this area is necessary. The cells concerned with triggering macrophage activation could be characterized more clearly by the use of anti-theta serum and other anti-sera to cell surface markers. It should also be noted that CMI may not be the sole means by which macrophages acquire increased bactericidal capacity. Mice which have been neonatally thymectomized (Takeya, Mori and Imaizumi, 1968) or thymectomized as adults, lethally irradiated and reconstituted with isogeneic bone marrow (Blanden and Langman, unpublished data) possess activated macrophages without the necessity for any deliberate infection to provoke it; furthermore.

6 398 R. V. BLANDEN AND C. A. MIMS these animals have a greatly depressed capacity for CMI responses (Miller and Osoba, 1967; Blanden and Langman, unpublished data). The importance of macrophage activation as a mechanism of resistance to viral infection is not known, and clear evidence may be difficult to acquire experimentally because of the complicating effects of other antiviral factors such as antibody and interferon. However, ectromeiia virus has been shown to be less successful at parasitizing the livers and spleens of mice with littoral macrophages activated by Listeria infection or graft-versus-host reactions (Blanden, 1971b). The phenomenon is also of possible significance where macrophages are involved in age-dependent resistance to herpes simplex virus (Johnson, 1964; Hirsch, Zisman and Allison, 1970). More insight into this problem should come when more is known about the means by which macrophages inactivate ingested viruses and other intracellular parasites. BLANDEN, R. V. (1968): 'Modification of macrophage function.' /. reticuloendothel. Soc, 5, 179. BLANDEN, R. V. (1970): 'Mechanisms of recovery from a generalized viral infection: mousepox. I. The effects of antithymocyte serum.' /. exp. Med., 132, BLANDEN, R. V. (1971a): 'Mechanisms of recovery from a generalized viral infection: mousepox. II. Passive transfer of recovery mechanisms with immune lymphoid cells.' /. exp. Med., 133, BLANDEN, R. V. (1971b): 'Mechanisms of recovery from a generalized viral infection: mousepox. III. Regression of infectious foci.' /. exp. Med., 133, BLANDEN, R. V., LEFFORD, M. J., and MACKANESS, C. B. (1969): 'The host response to Calmette-Guerin bacillus infection in mice.' /. exp. Med., 129, BLANDEN, R. V., MACKANESS, C. B., and COLLINS, F. M. (1966): 'Mechanisms of acquired resistance in mouse typhoid.' /. exp. Med., 124, 585. CoDAL, T., REES, R. J. W., and LAMVIK, J. O. (1971): 'Lymphocyte-mediated modification of blood-derived macrophage function in vitro; inhibition of growth of intracellular mycobacteria with lymphokines.' Clin. exp. Immunol, 8, 625. HIRSCH, M. S., ZISMAN, B., and ALLISON, A. C. (1970): 'Macrophages and agedependent resistance to herpes simplex virus in mice.' /. Immun., 104, REFERENCES. JOHNSON, R. T. (1964): 'The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of resistance with age.' /. exp. Med., 120, 359. MACKANESS, C. B., and BLANDEN, R. V. (1967): 'Cellular immunity.' Frog. Allergy, 11, 89. MILLER, J. F. A. P., and OSOBA, D. (1967): 'Current concepts of the immunological function of the thymus.' Physioi. Rev., 47, 437. MiMS, C. A. (1964): 'Aspects of the pathogenesis of virus diseases.'bact. Reu., 28,30. MOONEY, J. J., and WAKSMAN, B. H. (1970): 'Activation of normal rabbit macrophage monolayers by supernatants of antigen-stimulated lymphocytes.' /. Immun., 105, NATHAN, C. F., KARNOVSKY, M. L., and DAVID, J. R. (1971): 'Alterations of macrophage functions by mediators from lymphocytes.' /. exp. Med., 133, PATTERSON, R. J., and YOUMANS, C. P. (1970): 'Demonstration in tissue culture of lymphocyte-mediated immunity to tuberculosis.' Infec. Immunity, 1, 600. SIMON, H. B., and SHEACREN, J. N. (1971): 'Cellular immunity in vitro. 1. Immunologically mediated enhancement of macrophage bactericidal capacity.' /. exp. Med., 133, TAKEYA, K., MORI, R., and IMAIZUMI, N. (1968): 'Suppressed multiplication of Listeria monocytogenes within macrophages derived from thymectomized mice.' Nature, 218, 1174.

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