INFECTION AND IMMUNITY, Nov. 1979, p. 422426 0019-9567/79/11-0422/05$02.00/0

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Ia Antigens in Serum During Different Murine Infections CHRISTOPHER R. PARISH,l* ROBERT R. FREEMAN,' IAN F. C. McKENZIE,2 CHRISTINA CHEERS,3 AND GERALD A. COLE4 Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T.,' Department of Medicine, Austin Hospital, Heidelberg, Victoria,2 and Department of Microbiology, University of Melbourne, Parkville, Victoria,3 Australia; and the Department of Epidemiology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland

212054 Received for publication 3 July 1979

There exists in the mouse a family of I-region-controlled (Ia) antigens which carbohydrate-defined determinants. These antigens appear in serum as glycolipids and seem to be actively secreted by antigen-activated T-cells. This paper describes the ability of selected viral, bacterial, and protozoal infections of mice to markedly alter the serum levels of these Ia antigens. All the infectious agents examined induced substantial augmentation or suppression of serum Ia concentrations or both. Lymphocytic choriomeningitis (LCM) virus first enhanced and then suppressed serum Ia levels during the course of acute infection. Enhancement occurred during the time of ongoing virus replication and splenic lymphoproliferation while suppression coincided with the peak of the cytotoxic T-cell response and virus clearance. Listeria monocytogenes infection induced a substantial reduction in Ia levels at a time just after marked depletion of T-cells in the spleen. In contrast, Brucella abortus caused a significant increase in Ia levels 7 days postinfection, which correlates with the appearance of peak numbers of bacteria in tissues. Finally, Plasmodium yoelii, a nonlethal malarial parasite which stimulates prolonged T-cell proliferation, augmented serum Ia levels, whereas P. berghei, a lethal parasite which tends to inhibit T-cell division, suppressed Ia secretion. Possible interpretations of these different results are presented. carry

The Ia antigens of the mouse are controlled by a cluster of genes located within the H-2 complex and represent a highly polymorphic group of alloantigens (5, 22). The functional significance of these Ia antigens is still uncertain, but there is a strong possibility that they are the product of Ir genes. Studies in a number of laboratories indicate that the La antigens are glycoprotein molecules with a molecular weight of 58,000, which can be dissociated into two nonidentical polypeptides with a molecular weight of 33,000 and 25,000 (4, 21). Furthermore, the antigenic determinants of these molecules appear to reside in their protein portion (4, 21). Recently, data have been reported which suggest that the I-region controls a second family of antigens, both in mice (11, 17, 20) and humans (20a), which have carbohydratedefined antigenic determinants. These Ia antigens are expressed on the surface of most Blymphocytes and a subpopulation of T-cells and they also are present in serum in substantial concentrations (16). They probably exist as glycolipids in serum and on cells and are clearly

distinct from the more conventional glycoprotein Ia antigens (17). The levels of the carbohydrate-defined Ia antigens in serum appear to be under T-cell control (12, 15). Furthermore, alloantigens (15) and mitogens (19) are potent stimulators of Ia secretion by T-cells, whereas other nonreplicating antigens are comparatively poor stimulators of Ia antigen production (19). In the light of these findings, a number of infectious agents were assessed for their ability to alter the concentrations of the Ia antigens normally found in the serum of mice.

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MATERIALS AND METHODS Mice. Inbred CBA/H and BALB/c mice of both sexes were used at 6 to 10 weeks of age for serum collection. In some cases BALB/c mice persistently infected with lymphocyte choriomeningitis (LCM) virus were used. These were obtained from a colony of congenitally infected carrier mice maintained at the John Curtin School of Medical Research, Canberra, Australia. Infectious agents. The Armstrong (E350) strain of LCM virus was used in this study. Stock virus

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consisted of a clarified 20% suspension of infected mouse brain stored in small portions at -70oC, one of which was used for each experiment. Brucella abortus 19, a smooth attenuated vaccine strain, was obtained from Commonwealth Serum Laboratories, Parkville, Victoria, Australia. Listeria monocytogenes was obtained originally from V. P. Ackerman and had a 50% lethal dose of 3 X 103 organisms for CBA/H mice. Both bacteria were maintained by weekly subculture on horse blood agar and were renewed from lyophilized stock after fewer than 50 subcultures. Plasmodium berghei K173 was donated by M. J. Howell, Australian National University, Canberra, Australia, and was maintained in mice by blood passage every 10 days. P. yoelii 17X was obtained from D. Walliker, Institute of Animal Genetics, Edinburgh, Scotland, and was stored at -70'C as a stabilate of infected mouse blood. Infection of mice. Adult BALB/c mice were inoculated intravenously (i.v.) with 0.2 ml of diluted stock LCM virus containing ca. 13,000 adult mouse intracerebral (i.c.) 50% lethal doses (LD50). Such mice all developed a self-limiting immunizing infection (3, 9). Newborn BALB/c mice less than 2 days of age were inoculated i.c. with 0.02 ml of diluted stock virus (ca. 5,000 adult mouse i.c. LD50) to produce persistent virus carriers (3). Adult CBA/H mice were injected i.v. with 0.2 ml containing either 5 x 105 viable B. abortus bacteria or 103 viable L. monocytogenes. BALB/c mice were infected i.v. (0.2 ml) with 10' P. berghei or P. yoelii parasitized erythrocytes. During malarial infections parasitaemia was measured by microscopic counting of parasitized erythrocytes in Giemsa-stained blood smears. Rabbit anti-Ia serum. A rabbit anti-mouse Ia serum, which has been previously characterized (16, 20), was used to detect Ia antigens in serum. The antiserum was prepared by immunizing rabbits with CBA/H whole mouse serum and extensively absorbing the resultant antiserum with dialyzed CBA/H serum (16). Extensive studies indicate that this antiserum reacts with a range of I-region controlled antigens which appear as glycolipids in serum (15-20). Direct testing and absorption studies (20) have shown that the antiserum reacts with antigenic specificities genetically resembling Ia antigens 1, 3, 7, 15, 17, 19, and 22. These specificities are all carried by CBA/H (H-2k) spleen cells, whereas antibodies to Ia antigens 7 and 15 react with BALB/c (H-2d) splenocytes. Assay of Ia antigen levels in serum. The relative amounts of Ia antigen in sera were determined by an antigen-antibody inhibition assay which has been reported previously (16, 19). This assay measured the ability of serial dilutions of Ia-containing mouse (CBA/H or BALB/c) serum to inhibit the binding of a constant amount of the rabbit anti-mouse Ia antibody to "target" splenic lymphocytes from mice of the same strain. Binding of the rabbit anti-mouse Ia antibody to target cells was detected by a resetting procedure, using sheep anti-rabbit immunoglobulincoated erythrocytes (20). Briefly, the inhibition assay was as follows. To 50 ul of a 1/100 dilution of the rabbit anti-Ia serum was added serial dilutions (50 ul) of serum, and the mixture was incubated overnight at 4°C. A 100-pl amount of spleen cells (2 x 107/ml in phosphate-buffered saline-10% fetal calf serum) was

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added to each tube, the tubes were incubated on ice for 30 min, and the cells were washed twice with medium. Then 0.5 ml of a 2% suspension of sheep erythrocytes coated, via CrC13, with sheep immunoglobulin G (IgG) specific for rabbit immunoglobulin was added (16, 20). The erythrocyte-lymphocyte mixture was pelleted by centrifugation to encourage rosette formation, the cell pellets were resuspended in their supernatants, and resetting lymphocytes were counted with the aid of a crystal violet staining solution (16, 20). Results were expressed as the inhibitory activity per milliliter of each mouse serum sample tested and represented the reciprocal of the serum dilution required for 50% inhibition of resetting. Both infected and uninfected, age-matched control mice were bled only once from either the retroorbital venous plexus or the tail vein. Assays were performed on either individual or pooled sera from groups of three to five mice after all sera from any given experiment had been collected. Accumulated sera were stored at -70°C until assayed.

RESULTS In a series of experiments, the ability of different infectious agents to modify existing serum levels of carbohydrate-defined Ia antigens was determined. The results obtained with each infection are presented below. LCM virus infections. During the course of self-limiting LCM infections produced in adult mice, there were substantial changes in the serum levels of Ia antigen (Fig. 1). Five days after virus inoculation there was an approximately 20fold increase in Ia levels, which at day 9 had declined to one-fifth the concentration found in 10240r

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FIG. 1. Ia antigen levels in the serum of adult BALB/c mice at various times after acute infection with LCM virus. Each point represents the inhibition titer of serum from an individual mouse. The day-O points represent the inhibition titers of nine agematched uninfected mice.

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serum of uninfected controls. By day 15 postin- occurring maximally (95% suppression) on day fection, serum Ta levels had returned to normal. 6. On the other hand, Brucella produced a fourA different pattern was seen in mice with fold augmentation of serum Ia concentration at established chronic carrier infections (Table 1). day 7 postinfection. In both infections La levels Sera from both types of carriers, i.e., animals had returned to normal by day 10. that acquired their infection vertically from inMalarial infections. Two malarial parasites fected mothers (3), as well as those infected were examined: P. berghei, which in mice proshortly after birth, contained substantially re- duces high parasitemias and is ultimately lethal, duced (ca. 10 to 20% of control values) Ia antigen and P. yoelii, which is nonlethal and causes a levels. transient, relatively low level, parasitemia (6). Brucella and Listeria infections. In mice, P. yoelii induced an increase in serum Ia levels both Brucella and Listeria bacteria multiply which peaked at about day 6 postinfection and preferentially within macrophages; however, then returned to normal background levels by Listeria produces an acute self-limiting infec- day 12 (Fig. 3). In contrast, serum Ia in P. tion, whereas Brucella infections remain chronic berghei-infected mice, after an initial elevation, (10). These two bacterial infections had very fell by day 9 to subnormal levels, which persisted different effects on serum Ta levels (Fig. 2). Lis- for as long as the animals survived. Although teria caused a significant depression of serum Ia not shown, after infection with either species of levels between days 3 and 8 postinfection, but Plasmodium, the increased levels of serum Ia that existed during the pre-patent period showed TABLE 1. Levels of Ia antigen in the serum of a considerable decline concomitant with rising different LCM virus carrier micea parasitemias. Ia inhibition titer/ml of serum BALB/c mice that DISCUSSION Individual mice

were:

Mean

60 40,80, 60 Congenital carriers 40 20,40, 60 Neonatal carriers 320 Uninfected controls 640, 320, 320, 160 aProduction of persistently infected carrier mice is described in the text. Carriers and uninfected controls were bled at 8 to 10 weeks of age.

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The data presented in this paper indicate that viral, bacterial, and protozoal infections can produce substantial enhancement or suppression of the concentrations of carbohydrate-defined Ia antigens in serum or both. Previous studies (12) have implicated a subclass of T-cells in Ia secretion. These were characterized as being Ly-1 +2and la-, a cell-surface phenotype that typifies 1280

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6 9 12 15 18 21 24 DAYS AFTER INFECTION FIG. 2. Effect of B. abortus (A) and L. monocytogenes (A) infections on serum Ia antigen levels in CBA/H mice at various times after infection. In the case of L. monocytogenes infection, at each time point serum was pooled from five infected mice, whereas with B. abortus infection, inhibition titers are expressed as the mean of three mice per time point + standard error of mean. 0

3

9 12 15 18 DAYS AFTER INFECTION FIG. 3. Effect of P. berghei (A) and P. yoelii (A) infections on serum Ia antigen levels in BALB/c mice at various times after infection. Results are expressed as mean inhibition titers of four mice per time point + standard error of mean. Dotted line represents inhibition titer of serum from age-matched uninfected 3

mice.

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T-cells having a helper function as well as those which mediate delayed-type hypersensitivity reactions (1, 24). Therefore, it is likely that serum Ia is an indicator of T-cell activation. On this basis, several comments can be made about the results obtained during each type of infection. The ability of LCM virus to induce first augmented and then depressed serum Ia levels (Fig. 1) correlates with other well-described parameters of the acute self-limiting infection produced when this agent is given extraneurally to adult mice. The augmentation seen on day 5 of infection corresponds to the time of ongoing virus replication (23) and cell proliferation in lymphoid tissue (9). Depression of serum Ia coincides with the peak of the cytotoxic T-cell response against virus-infected cells (9) and also with the time when macrophages, in which LCM virus replicates preferentially, are rendered markedly dysfunctional (7). It therefore seems that immune-mediated destruction of macrophages, which are probably essential for activation of Iasecreting T-cells, may be the primary mechanism of Ia depression. The abnormally low serum levels of La in both types of LCM virus-carrier mice is more difficult to explain. Previous studies have not revealed any evidence of macrophage dysfunction in carrier mice (7), although the tendency of such animals to develop circulating virus-antibody complexes (14) may underlie their apparent low level of T-cell activation. Because LCM viruscarrier mice, by definition, first experience infection at a time when their T-cell system is either immature or suppressed (3, 9), it is possible that the continued presence of free or completed virus causes a lasting, albeit subtle, T-cell defect which results in Ia suppression. The two bacterial infections examined, listeriosis and brucellosis, produced very different effects on serum Ia levels (Fig. 2). Although both agents multiply preferentially within macrophages of the spleen and liver, they present contrasting histopathological pictures. Listeria infection causes a marked depletion of the Tcell-dependent areas of the spleen, accompanied by a temporary reduction in numbers of splenic Thy-1+ lymphocytes that is most marked after 3 to 4 days followed by T-cell repopulation by' days 6 to 8 (T. Mandel and C. Cheers, in preparation). Thus T-cell depletion immediately precedes the striking suppression of Ia synthesis seen during infection. Interestingly, both previously reported macrophage activation and the appearance of Listeria-specific delayed type hypersensitivity (13) parallel the time course of the La suppression seen in the present study. The results obtained with B. abortus are more difficult to interpret. Brucella, in contrast to

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Listeria, caused a substantial increase in Ia levels by day 7 postinfection. Brucella infection of mice does not cause the T-cell depletion seen in listeriosis but, instead, results in a gross influx of macrophages to the spleen (C. Riglar and C. Cheers, Cell. Immunol., in press). The day-7 peak in Ia antigen levels during brucellosis corresponds with the beginning of this macrophage influx as well as the appearance of maximum numbers of bacteria and the onset of cell-mediated immunity (2, 10). However, peak Brucella specific immune responses are not reached until week 3 of infection when Ia levels have returned to normal. It is possible that the apparent stimulation of Ia secretion is caused by the lipopolysaccharide of Brucella because bacterial polysaccharides are known to be potent stimulators of Ia production (19). Finally, the results obtained with the two rodent strains of Plasmodium correlate well with the opposite effects these parasites have on Tcells. Thus, P. yoelii, which stimulates prolonged T-cell proliferation (6, 8), also augmented serum Ia levels, whereas P. berghei, which tends to suppress T-cell division (6, 8), caused a suppression of Ia secretion (Fig. 3). It could be argued that in some cases the variations in serum Ia levels during infection could be produced by the organisms or their products interfering with the inhibition assay. This seems unlikely, however, because the inhibitory substance in serum from LCM-infected and Brucella-infected mice was found to be a dialyzable molecule, i.e., probably glycolipid. Furthermore, selected sera from LCM, Brucella, and Listeria infections gave similar results when used to inhibit mouse anti-Ia sera. Collectively, the data presented in this paper suggest that the levels of carbohydrate-defined Ia antigens in serum represent a sensitive marker for certain types of immune responses to infectious agents, particularly during the stage of ongoing macrophage-T-cell interaction. Thus, suppression of Ia levels in serum appears to correlate with either macrophage dysfunction (e.g., LCM virus infection) or T-cell depletion (e.g., Listeria and P. berghei infections), whereas stimulation of Ia secretion correlates with T-cell proliferation (e.g., LCM virus and P. yoelii infections). However, a more precise interpretation of our findings will be possible when the functional significance of these Ia antigens is firmly established. ACKNOWLEDGMENTS We acknowledge the technical assistance of Aira Chilcott. I. F. C. McKenzie acknowledges research grants from the NHMRC (Australia) and Australian Tobacco Research Foundation and Public Health Service grant CA-21224 from the National Institutes of Health. C.C. was supported by a Na-

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tional Health and Medicine Research Council (Australia) grant. G.A.C. was supported by U.S. Public Health Services grant NS-11286 from the National Institutes of Health and a Josiah Macy Foundation Fellowship.

UITERATURE CITED 1. Cantor, H., and E. A. Boyse. 1975. Functional classes of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly' cells in the generation of killer activity. J. Exp. Med. 141:1390-1399. 2. Cheers, C., and F. Pagram. 1979. Macrophage activation during experimental murine brucellosis: a basis for chronic infection. Infect. Immun. 23:197-205. 3. Cole, G. A., and N. Nathanson. 1974. Lymphocytic choriomeningitis virus. Pathogenesis. Prog. Med. Virol. 18:94-110. 4. Cullen, S. F., J. H. Freed, and S. G. Nathenson. 1976. Structural and serological properties of murine Ia alloantigens. Transplant. Rev. 30:236-270. 5. David, C. S. 1976. Serological and genetic aspects of murine Ia antigens. Transplant. Rev. 30:299-330. 6. Freeman, R. R., and C. R. Parish. 1978. Spleen cell changes during fatal and self-limiting malarial infections of mice. Immunology 35:479-484. 7. Jacobs, R. P., and G. A. Cole. 1976. -Lymphocytic choriomeningitis virus-induced immunosuppression: a virus-induced macrophage defect. J. Immunol. 117:10041009. 8. Jayawardena, A. N., G. A. T. Targett, E. Leuchars, R. L Carter, M. J. Doenhoff, and A. J. S. Davies. 1975. T-cell activation in murine malaria. Nature (London) 258:149-151. 9. Johnson, E. D., and G. A. Cole. 1975. Functional heterogeneity of lymphocytic choriomeningitis virus-specific T lymphocytes. I. Identification of effector and memory subsets. J. Exp. Med. 141:866-881. 10. Mackaness, G. B. 1964. The immunological basis of acquired cellular resistance. J. Exp. Med. 120:105-120. 11. McKenzie, I. F. C., A. E. Clarke, and C. R. Parish. 1977. Ia antigenic specificities are oligosaccharide in nature: Hapten inhibition studies. J. Exp. Med. 145: 1039-1053. 12. McKenzie, I. F. C., and C. R. Parish. 1976. Secretion of Ia antigens by a subpopulation of T cells which are Ly1+, Ly-2- and Ia-. J. Exp. Med. 144:847-851. 13. North, R. J. 1969. Cellular kinetics associated with the

INFECT. IMMUN. development of acquired cellular resistance. J. Exp. Med. 130:299-311. 14. Oldstone, M. B. A., and F. J. Dixon. 1971. Immune complex disease in chronic viral infections. J. Exp. Med. 134:32s-40s. 15. Parish, C. R., A. B. Chilcott, and L F. C. McKenzie. 1976. Low molecular-weight Ia antigens in normal mouse serum. II. Demonstration of their T-cell origin. Immunogenetics 3:129-137. 16. Parish, C. R., A. B. Chilcott, and L. F. C. McKenzie. 1976. Low molecular-weight Ia antigens in normal mouse serum. I. Detection and production of a xenogeneic antiserum. Immunogenetics 3:113-128. 17. Parish, C. R., T. J. Higgins, and I. F. C. McKenzie. 1978. Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: evidence for two classes of Ia antigens. Immunogenetics 6:343-354. 18. Parish, C. R., D. C. Jackson, and L F. C. McKenzie. 1976. Low molecular-weight Ia antigens in normal mouse serum. III. Isolation and partial chemical characterization. Immunogenetics 3:455463. 19. Parish, C. R., and I. F. C. McKenzie. 1977. Mitogens and T-independent antigens stimulate T lymphocytes to secrete Ia antigens. Cell. Immunol. 33:134-144. 20. Parish, C. R., and L. F. C. McKenzie. 1978. A detailed serological analysis of a xenogeneic anti-Ia serum. Immunogenetics 6:183-196. 20a.Sandrin, M. S., H. A. Vaughan, L. F. C. McKenzie, B. D. Tait, and C. R. Parish. 1979. The human Ia system: definition and characterization by xenogenetic antisera. Immunogenetics 8:185-200. 21. Schwartz, B. D., and S. E. Cullen. 1978. Chemical characteristics of Ia antigens. Springer Seminars Immunopathol. 1:85-110. 22. Shreffler, D. C., and C. S. David. 1975. The H-2 major histocompatibility complex and the I immune response region. Genetic variation, function and organization. Adv. Immunol. 20:125-187. 23. Silberman, S. L, R. P. Jacobs, and G. A. Cole. 1978. Mechanisms of hemopoietic and immunological dysfunction induced by lymphocytic choriomeningitis virus. Infect. Immun. 10:533-539. 24. Vadas, M. A., J. F. A. P. Miller, L. F. C. McKenzie, S. E. Chism, F.-W. Shen, E. A. Boyse, J. R. Gamble, and A. M. Whitelaw. 1976. Ly and Ia antigen phenotypes of T cells involved in delayed-type hypersensitivity and in suppression. J. Exp. Med. 144:10-19.