Leukemia ResearchVol. 3, No. 2, pp. 67-74. © Pergamon Press Ltd., 1979.Printedin Great Britain.
0145-2126/79/0401-0067$02.00/0
CHANGES OF SERUM THYMIC FACTOR FRIEND LEUKEMIA VIRUS-INFECTED
LEVELS MICE
IN
ENRICO GARACI, VERA DEL GOBBO, LAURA SANTUCCI Institute of Microbiology, Rome University, Italy GIOVANNI BATTISTAROSSI Laboratory of Bacterial and Viral Diseases, Istituto Superiore di Sanit/~, Rome, Italy a n d CRISTINA RINALDI-GARACI I1 Chair of General Pathology, Rome University, Italy (Received 9 August 1978. In revisedform 18 September 1978. Accepted 9 October 1978) Abstraet--Sera and spleen cells from DBA/2 and BALB/c mice were sampled at various time intervals after infection with Friend leukemia virus (FLV, polyeythemic strain) and assayed for the presence of serum thymic factor (STF) and theta-positive cells, respectively. A pronounced and dose-dependent decrease of STF levels was observed in DBA/2 mice as early as 48 h after infection; STF levels were decreased in BALB/c mice, too, but the effect was less marked and long-lived than in DBA/2 mice. In close parallelism to the fall of STF levels in the sera, theta-positive cells were also found to be markedly decreased in the spleens of DBA/2 mice and, to a lesser extent, in BALB/c mice. Thetapositive cells were assayed by measuring the sensitivity to azathioprine of spleen rosette-forming cells. Both the fall of STF levels and the decrease of theta-positive spleen cells do not persist longer than 4 weeks and are not, therefore, related to the state of overt leukemia. Conceivably, though, these changes, that appear to be virus-related, may influence the early fate of FLV-transformed cells by lowering some thymus-dependent functions which may be relevant to the immunological surveillance. Key words: Thymic Factor, Friend leukemia virus, cell-mediated immunity, theta-positive cells, azathioprine, leukemogenesis
INTRODUCTION PREUMINARY evidence has been provided pointing to possible anti-tumor effects of biologically active thymic factors in some animal tumor models [20, 21, 32, 34]. In view of the well-known regulatory effects of thymic factors on cell-mediated immunity, these data may provide some support to the claim of a relationship between tumor onset and immunosuppression. The Friend leukemia virus (FLV) causes a drastic depression of the humoral immune response [9, 10, 13, 28]. A lot of data suggests that several parameters of cell-mediated immune functions are impaired by FLV infection [16, 26, 27] but contradicting evidence is also available [6, 8, 15]. In view of the prominent role of the thymus [24] or of its hormonal products in the immune response and more specifically in the maturation of T cells and regulation of their functions [7, 25], we have studied the effects of FLV infection on the levels of serum thymic factor (STF) (a thymus-derived hormone isolated from human and murine sera [3]) and of thetapositive spleen ceils as detected by the azathioprine (AZ) inhibition test. Correspondence to: Professor Enrico Garaci, Institute of Microbiology, Piazzale delle Scienze, Rome University, 00100 Rome, Italy. Abbreviations: FLV, Friend leukemia virus; STF, Serum thymic factor; AZ, Azathioprine; Tx, thymectomized; sRFC, Spleen rosette-forming cells. 67
68
ENRICO GARACIet aL
In this c o n t r i b u t i o n we r e p o r t p r e l i m i n a r y data indicating that the levels o f b o t h S T F an d theta-positive spleen cells are decreased in the early stages o f viral infection a n d / o r leukemic t r a n s f o r m a t i o n . T h e r e is a direct relationship between the m a g n i t u d e o f these effects a n d the virus dosage.
MATERIALS AND
METHODS
Mice Five to six weeks old male DBA/2 and BALB[c n-6ce (generously provided by Dr. V. Monaco, CNENCasaccia, Rome) were used to produce virus stocks and to test their effects on the immunological parameters studied. Random-bred Swiss mice (obtained from the same source) aged 6-11 weeks, were employed for virus titration.
Virus The N-tropic polycythemic strain of FLV (routinely passaged in our laboratory) has been used throughout this study. Twenty per cent cell-free homogenates of leukemic spleens were prepared according to Friend [17] and titrated in Swiss mice for induction of splenomegaly. Titration experiments were terminated 90 days after virus inoculation. The two virus stocks used had a titer, calculated according to the Reed and Muench formula, of 10"'a and 105"°- LDso/0.2 ml. Both virus stocks were free of lactic dehydrogenase virus [31]. The standard virus inoculum was 0.2 ml/mouse, intraperitoneally.
Evaluation o f the level o f aerum thymic factor STF determinations were performed according to the method of Bach and Dardenne [2]. The principle of the assay consists of the induction of theta antigen on theta-negative T cell precursors after incubation with circulating thymic hormone. For this purpose, either AZ or anti-theta serum may be used. Both these substances, at low concentrations, are capable of inhibiting, with absolutely identical results [4], the formation of rosettes by theta-positive spleen rosette-forming cells (sRFC). In fact sRFC from adult thymectomized mice show no sensitivity to them, whereas sRFC from normal mice do and the sensitivity of the former's returns after incubation with thymic hormone. For practical reasons, AZ was preferred to anti-theta serum. C57BL/6 mice were thymectomized (Tx) at the age of 8-10 weeks. Ten to fifteen days later their spleen cells were collected, dissociated, washed twice and incubated with variable log2 dilutions of test sera at a concentration of 15 × 10~/ml in Hanks medium. Before incubation, test sera were ultrafiltrated by diafiltration on Amicon UM-10 in order to exclude molecules with MW over 10,000 Daltons. AZ was then added at low concentration (10 ~tg/ml). This concentration was selected as the one capable of inhibiting sRFC in normal mice, while much higher concentrations of AZ (50 ~tg/ml or more) are also capable of inhibiting sRFC from adult "Ix mice. In the absence of STF, rosette formation is not inhibited by the AZ concentration selected, while in its presence AZ inhibits rosette formation as it would do, at the same concentration, for sRFC from normal mice. After 75 rain incubation at 37°C, sheep red blood cells were added at a concentration of 48 × 10~/ml and the cell suspension was centrifuged at 4°C and re-suspended gently. Rosettes were read on a hemocytometer. The highest serum dilution of the tested sample which was still able to restore AZ sensitivity (i.e. to provoke 50% inhibition of RFC) was taken as the active concentration of circulating thymic factor and was expressed as titer of the serum.
Evaluation of the presence of theta-positive splenic cells by the azathioprine inhibition test The presence of theta-positive cells in the spleens of infected mice was ascertained by the technique for the rosette inhibition test described by Bach and Dardenne [2]. Briefly, the test is similar to that previously described, except that increasing doses of AZ were added so as to achieve a minimal inhibitory concentration defined as the quantity that reduces by greater than 50 % the number of sRFC counted in a given field, as compared to controls.
RESULTS
Effects o f E L V inoculation on the levels o f S T F T h e levels o f S T F detected (at v a r i o u s time intervals after virus inoculation) in D B A / 2 mice i n o c u l a t e d either with heat-inactivated o r with v ar i o u s doses o f live F L V , are sh o w n in Fig. 1.
Serum thymic factor in Friend virus-infected mice
69
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days after FLV administration FIG. 4. Same protocol as in Fig. 3. Symbols as in Fig. 1.
DISCUSSION The data presented in this paper clearly show that the administration of FLV to DBA/2 mice results in a pronounced decrease of the levels of STF and theta-positive splenic cells. These changes are detectable already on day 1 after virus inoculation, remain evident for about 4 weeks and tend to revert to normal values later on, even in animals with overt leukemia. Interestingly, the levels of thetaopositive cells of mice given undiluted FLV remained low even at 60 days after infection, whereas, with 10-1 and 10-2 FLV dilutions, their values bounced back to normality. One possible explanation is that the FLV-induced drastic decrease of STF levels, which the spleen theta-positive cells are very much dependent upon, has profoundly affected the recovery of these cells. Alternatively, one may envision a direct action of FLV on the splenic cells plasma membrane that may render them less sensitive to AZ. Maseio and Ceglowski [23] found a decreased sensitivity of mouse spleen cells, infected by FLV, to anti-theta serum. The two hypotheses are not mutually exclusive. The close parallelism of the kinetics of the two effects may indicate either an additive or a synergistic effect caused by lack of STF and FLV infection. In fact, the decrease of AZ spleen sensitivity caused by thymectomy [1] is neither as early nor as pronounced as that observed in our studies. The observed changes in STF levels and AZ sensitivity are dose-dependent, suggesting that they are related to virus replication. Additional evidence for this relationship is provided by the data observed in BALB/c mice given the same FLV preparations. BALB/c mice (Fv-1 b/b) are partially resistant to the replication of N-tropic strains of FLV. In keeping with data from the literature, our stocks of FLV showed a 3-1oglo reduction of titer when tested in BALB/c mice (data not shown). Under these conditions, both STF levels and AZ sensitivity were still reduced, in a dose-dependent fashion, by FLV inoculation but at a much lesser extent than in DBA/2 mice (Fv-ln/n). This indicates that a genotype partially restrictive for FLV replication is also restrictive for the observed modulation of the parameters under study.
72
ENRICO GARACIet al.
The mechanism by which FLV infection causes the observed drastic decrease of STF levels remains to be determined. A possible explanation could be provided by a direct effect of FLV on thymic epithelial ceils that actually secrete biologically active thymic products. We have, in fact, shown in various experimental conditions that the fall of STF levels is associated with profound alterations of these cells [14, 18, 19]. Preliminary observations, made with the electron microscope on day 2 and 16 after virus inoculation, do not appear to show widespread FLV replication in the epithelial cells. Still, negative data are never definitive because of the well known "sampling" error built in the ultrastructural studies and more work, therefore, is needed to resolve this issue. Another explanation may be as follows: the normal STF levels differ in DBA/2 and BALB/c mice. This suggests the possibility that mice with high constitutive STF levels may be more responsive to the "virus effect". The data described in this study do not allow us to draw an obvious correlation between the decrease of STF values and FLV leukemogenesis. As a matter of fact, overtly leukemic mice (more than 30 days after infection) did not have decreased STF levels and, moreover, had low levels of theta-positive spleen cells only occasionally (compare data in mice given undiluted and 10-1 FLV dilution). Yet, the peculiar kinetics of both effects (early and pronounced fall of STF and theta-positive cells in DBA/2 mice and to a lesser extent, in BALB/c mice) is suggestive of a possible pathogenetical relationship between these changes and the onset of FLV-induced leukemogenesis. One might ask the question whether the early fall in STF levels may play a role in the establishment of FLV infection and/or transformation of its target cells. In other words, the lack of STF soon after infection with FLV may cause an overall derangement of the immune system which may become less ready to fight and destroy the rapidly emerging leukemic cell population. The leukemic cells do possess new viral antigens and do also express hemopoietic histocompatibility antigens, that are not expressed in the corresponding normal hemopoietic cells [11, 12, 22, 29, 30]. The strength of the immune response against all these antigens may be very instrumental in determining the fate of the newly made leukemic cells. On the other hand, it is known that STF activity in man is decreased in several T-cell deficiencies and in advanced age [4]. In mice, STF activity decreases with age [5]; its administration, moreover, enhanced the ability of mice and rats to reject tumor grafts [20, 21, 34]. The conflicting evidence on the impairment of T-cell functions in FLV-infected mice [6, 8, 15, 16, 26, 2"1], may be accounted for by the fact that low STF titers do not necessarily induce an easily detectable defect of cell-mediated immunity. In fact, in adult thymectomized mice, the immediate fall of STF following removal of the gland is not accompanied by the simultaneous appearance of immunological changes thanks to the persistence of long-lived T-lymphocytes. Yet, some impairment of T-cell functions appears later [33]. Finally, it should be mentioned that the polycythemic strain of FLV, used in these studies, consists of a mixture of at least two viruses, i.e. the spleen focus-forming virus and the leukemia virus. The data presented here do not allow to attribute the observed changes to either one of the viruses. Studies in progress with the anemic strain of FLV (which lacks the focus-forming component, but induces an erythroleukemia indistinguishable from that induced by the polycythemic strain of FLV) will probably help to elucidate this point. Acknowledgements--The authors are indebted to Mr. G. Bagella for skilful technical assistance, and to Dr. V. Monaco for generous gifts of mice. This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CTB.77.01355.04 and Progetto Finalizzato Virus CT.77.00304.84).
Serum thymic factor in Friend virus-infected mice
73
REFERENCES 1. BACHJ. F., DARDENNEM. & DAVIESA. J. S. (1971) Early effect of adult thymectomy. Nature New Biol. 231, 110. 2. BACHJ. F. & DARDENNEM. (1973) Studies on thymus products--II. Demonstration and characterization of a circulating thymic hormone. Immunology 25, 353. 3. BACHJ. F., DAmaENNEM. & BACH M. A. (1973) Demonstration of a circulating thymic hormone in mouse and in man. Transplantation Prec. 5, 523. 4. BACHJ. F., DARDENNEM., PtmAUJ. M. & BACHM. A. (1975) Isolation, biochemical characteristics and biological activity of a circulating thymic hormone in the mouse and in the human. Ann. N.Y. Acad. Sci. 249, 186. 5. BACHJ. F. & CARNAUDC. (1976) Thymic factors. Prog. Allergy 21, 342. 6. BAINBRIDGED. R. & BENDINELLIM. (1972) Circulation of lymphoid cells in mice infected with Friend leukemia virus. J. natl. Cancer Inst. 46, 773. 7. BEKKUMVAND. W. (1975) The Biological Activity o f Thymic Hormones. Kooyker Scientific Publications, Rotterdam. 8. B~rNL~r M. & STE~Vr.SR. A. (1970) Immunocompetent cell functions in mice infected with Friend leukemia virus. J. Natl. Cancer Inst. 44, 1107. 9. C-'~LOWSKIW. S. & FRIEDtC,AN H. (1967) Suppression of the primary plaque response of mice following infection with Friend disease virus. Prec. Soc. exp. Biol. Med. 126, 662. 10. CEGLOWSKIW. S. & FRIEDMANH. (1968) Immunosuppression by leukemia virns--I. Effect of Friend disease virus on cellular and humeral hemolysin responses of mice to a primary immunization with sheep erythrocytes. J. Immunol. 101, 594. 11, CL~KOWICZG. & RossI G. B. (1972) Hybrid resistance to parental DBA/2 grafts: independence from the H-2 locus--L Studies with normal hematopoietic cells. J. natl. Cancer Inst. 48, 131. 12. Ct~KowIcz G., RossI G. B., HADDADJ. R. & FRIEND C. (1972) Hybrid resistance to parental DBA/2 grafts: independence from the H-2 locus--II. Studies with Friend virus-induced leukemia cells. J. natl. Cancer Inst. 48, 997. 13. DENT P. B. (1972) Immunodepression by oncogenic viruses. Progr. reed. Virol. 14, 1. 14. D~ACZmNKOW. & GARACIE. (1976) Dark reticular epithelial cells of the thymus as the primary target of heterologous anti-lymphocyte serum in BALB/c mice. Cii#. lmmun, lmmunopath. 6, 213. 15. DRACOTrB. N., WEDDERBURNN. & DOENHOFFM. J. (1977) The immunodepressive effect of Friend virus~IH. Effects on spleen T cells. Immunology 33, 573. 16. FRL~J~MANH., MELNICK H., MU..lm L. & CEOLOWSKIW. S. (1973) Depressed allograft immunity in leukemia virus infected mice. Transplantation Prec. 5, 981. 17. FRIEND C. (1957) Cell-free transmission in adult Swiss mice of a disease having the character of a leukemia. J. exp. Med. 105, 307. 18. GARACIE., DEL GOBBO V. & RINALDI-GARACIC. (1976) Effects of antithymic reticuloepithelial cells serum on the levels of circulating thymic factor and on the sensitivity to azathioprine of spleen spontaneous rosette-forming cells. Experientia 32, 1475. 19. GARACIE., PECCIG., RINALDI-GARACIC., DEL GOBBOV. & TONmTn G. (1978) Ultrastructural and functional changes of the mouse thymus following treatment with an antiserum specific for thymic epithelial cells. Clin. Immun. Immunopath. 11, 157. 20. IKEHARAS., HAMASHIMAY. & MASUDAT. (1975) Immunological restoration of both thymectomized and athymic nude mice by a thymus factor. Nature 258, 335. 21. KHAW B. A. & RULE A. H. (1973) Immunotherapy of the Dunning leukemia with thymus extracts. Br. J. Cancer 28, 288. 22. KUMARV., CARUSOT. & BENNE'I7M. (1976) Mechanisms of genetic resistance to Friend virus leukemia in mice--III. Susceptibility of mitogen-responsive lymphocytes mediated by T cells. J. exp. Med. 143, 728. 23. MASCIOA. A. & CEGLOWSKIW. S. (1975) Friend leukemia virus-induced suppression of lymphocyte mediated cytotoxicity. Fed. Prec. 34, 973. 24. MILLERJ. F. A. P. & OSOBAD. (1963) Role of the thymus in the origin of immunological competence. In Wolstenholme and Knight Ciba Found. Study Group. Nature and Origin oflmmunologically Competent Cells, p. 62. Churchill, London. 25. MILLERJ. F. A. P. (1975) T-cell regulation of immune responsiveness. Ann. N. ]I. Acad. Sci. 249, 9. 26. MORTENSENR. F., CE~LOWSKIW. S. & FRIr~MAN H. (1973) Leukemia virus-induced immunosuppression--IX. Depression of delayed hypersensitivity and MIF production after infection of mice with Friend leukemia virus. J. Immunol. 111, 1810. 27. MORT~NSENR. F., C'E~LOWSKIW. S. & FRIEDMANH. (1974) Leukemia virus-induced intrnunosuppression--X. Depression of T cell-mediated cytotoxicity after infection of mice with Friend leukemia virus. J. lmmunol. 112, 2077. 28. OLD L. J., CLARKED. A., BENACEe.RAFB. & GOLDSMITHM. (1960) The reticuloendothelial system and the neoplastic process. Ann. N. Y. Acad. Sci. 88, 264. 29. RossI G. B., CUDKOWlCZG. & FRIEND C. (1970) Evidence for transformation of spleen ceils in vitro one day after infection of mice with Friend leukemia virus. Autonomous growth potential and expression of hybrid resistance genes. J. exp. Med. 131, 765.
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ENRICO GARACl et al.
30. RossI G. B., CUDKOWICZ G. & FRIEND C. (1973) Transformation of spleen cells three hours after infection in vivo with Friend leukemia virus, d. natl. Cancer lltst. 50, 249. 31. RowsoN K. E. K. & MAnY B. W. (1975) Lactic Dehydrogenase Virus. Virology Monographs, Springer-. Verlag, Wien. 32. SERROUB., RF2¢~T., SENELARR., DELORB., DtmoIS J. B. & THmRRYC. (1975) T-lymphocyte maturation and antitumoral effect of a thymic extract obtained from a stimulated model. Ann. N. Y. Acad. Sci. 249, 328. 33. TI-rtmMANG. B. & GOLDSTEIr~A. L. (1975) The role of thymosin in lymphocyte maturation. Boll. 1st. sieroter. Milan. 54, 203. 34. ZXSBLA'rrM., GOLDSTEINA. L., LILLY F. & WI-IITEA. (1970) Acceleration by thymosin of the development of resistance to murine sarcoma virus-induced in mice. Proc. natl. Acad. Sci. U.S.A. 66, 1170.
0145-2126/79/0401-0067$02.00/0
CHANGES OF SERUM THYMIC FACTOR FRIEND LEUKEMIA VIRUS-INFECTED
LEVELS MICE
IN
ENRICO GARACI, VERA DEL GOBBO, LAURA SANTUCCI Institute of Microbiology, Rome University, Italy GIOVANNI BATTISTAROSSI Laboratory of Bacterial and Viral Diseases, Istituto Superiore di Sanit/~, Rome, Italy a n d CRISTINA RINALDI-GARACI I1 Chair of General Pathology, Rome University, Italy (Received 9 August 1978. In revisedform 18 September 1978. Accepted 9 October 1978) Abstraet--Sera and spleen cells from DBA/2 and BALB/c mice were sampled at various time intervals after infection with Friend leukemia virus (FLV, polyeythemic strain) and assayed for the presence of serum thymic factor (STF) and theta-positive cells, respectively. A pronounced and dose-dependent decrease of STF levels was observed in DBA/2 mice as early as 48 h after infection; STF levels were decreased in BALB/c mice, too, but the effect was less marked and long-lived than in DBA/2 mice. In close parallelism to the fall of STF levels in the sera, theta-positive cells were also found to be markedly decreased in the spleens of DBA/2 mice and, to a lesser extent, in BALB/c mice. Thetapositive cells were assayed by measuring the sensitivity to azathioprine of spleen rosette-forming cells. Both the fall of STF levels and the decrease of theta-positive spleen cells do not persist longer than 4 weeks and are not, therefore, related to the state of overt leukemia. Conceivably, though, these changes, that appear to be virus-related, may influence the early fate of FLV-transformed cells by lowering some thymus-dependent functions which may be relevant to the immunological surveillance. Key words: Thymic Factor, Friend leukemia virus, cell-mediated immunity, theta-positive cells, azathioprine, leukemogenesis
INTRODUCTION PREUMINARY evidence has been provided pointing to possible anti-tumor effects of biologically active thymic factors in some animal tumor models [20, 21, 32, 34]. In view of the well-known regulatory effects of thymic factors on cell-mediated immunity, these data may provide some support to the claim of a relationship between tumor onset and immunosuppression. The Friend leukemia virus (FLV) causes a drastic depression of the humoral immune response [9, 10, 13, 28]. A lot of data suggests that several parameters of cell-mediated immune functions are impaired by FLV infection [16, 26, 27] but contradicting evidence is also available [6, 8, 15]. In view of the prominent role of the thymus [24] or of its hormonal products in the immune response and more specifically in the maturation of T cells and regulation of their functions [7, 25], we have studied the effects of FLV infection on the levels of serum thymic factor (STF) (a thymus-derived hormone isolated from human and murine sera [3]) and of thetapositive spleen ceils as detected by the azathioprine (AZ) inhibition test. Correspondence to: Professor Enrico Garaci, Institute of Microbiology, Piazzale delle Scienze, Rome University, 00100 Rome, Italy. Abbreviations: FLV, Friend leukemia virus; STF, Serum thymic factor; AZ, Azathioprine; Tx, thymectomized; sRFC, Spleen rosette-forming cells. 67
68
ENRICO GARACIet aL
In this c o n t r i b u t i o n we r e p o r t p r e l i m i n a r y data indicating that the levels o f b o t h S T F an d theta-positive spleen cells are decreased in the early stages o f viral infection a n d / o r leukemic t r a n s f o r m a t i o n . T h e r e is a direct relationship between the m a g n i t u d e o f these effects a n d the virus dosage.
MATERIALS AND
METHODS
Mice Five to six weeks old male DBA/2 and BALB[c n-6ce (generously provided by Dr. V. Monaco, CNENCasaccia, Rome) were used to produce virus stocks and to test their effects on the immunological parameters studied. Random-bred Swiss mice (obtained from the same source) aged 6-11 weeks, were employed for virus titration.
Virus The N-tropic polycythemic strain of FLV (routinely passaged in our laboratory) has been used throughout this study. Twenty per cent cell-free homogenates of leukemic spleens were prepared according to Friend [17] and titrated in Swiss mice for induction of splenomegaly. Titration experiments were terminated 90 days after virus inoculation. The two virus stocks used had a titer, calculated according to the Reed and Muench formula, of 10"'a and 105"°- LDso/0.2 ml. Both virus stocks were free of lactic dehydrogenase virus [31]. The standard virus inoculum was 0.2 ml/mouse, intraperitoneally.
Evaluation o f the level o f aerum thymic factor STF determinations were performed according to the method of Bach and Dardenne [2]. The principle of the assay consists of the induction of theta antigen on theta-negative T cell precursors after incubation with circulating thymic hormone. For this purpose, either AZ or anti-theta serum may be used. Both these substances, at low concentrations, are capable of inhibiting, with absolutely identical results [4], the formation of rosettes by theta-positive spleen rosette-forming cells (sRFC). In fact sRFC from adult thymectomized mice show no sensitivity to them, whereas sRFC from normal mice do and the sensitivity of the former's returns after incubation with thymic hormone. For practical reasons, AZ was preferred to anti-theta serum. C57BL/6 mice were thymectomized (Tx) at the age of 8-10 weeks. Ten to fifteen days later their spleen cells were collected, dissociated, washed twice and incubated with variable log2 dilutions of test sera at a concentration of 15 × 10~/ml in Hanks medium. Before incubation, test sera were ultrafiltrated by diafiltration on Amicon UM-10 in order to exclude molecules with MW over 10,000 Daltons. AZ was then added at low concentration (10 ~tg/ml). This concentration was selected as the one capable of inhibiting sRFC in normal mice, while much higher concentrations of AZ (50 ~tg/ml or more) are also capable of inhibiting sRFC from adult "Ix mice. In the absence of STF, rosette formation is not inhibited by the AZ concentration selected, while in its presence AZ inhibits rosette formation as it would do, at the same concentration, for sRFC from normal mice. After 75 rain incubation at 37°C, sheep red blood cells were added at a concentration of 48 × 10~/ml and the cell suspension was centrifuged at 4°C and re-suspended gently. Rosettes were read on a hemocytometer. The highest serum dilution of the tested sample which was still able to restore AZ sensitivity (i.e. to provoke 50% inhibition of RFC) was taken as the active concentration of circulating thymic factor and was expressed as titer of the serum.
Evaluation of the presence of theta-positive splenic cells by the azathioprine inhibition test The presence of theta-positive cells in the spleens of infected mice was ascertained by the technique for the rosette inhibition test described by Bach and Dardenne [2]. Briefly, the test is similar to that previously described, except that increasing doses of AZ were added so as to achieve a minimal inhibitory concentration defined as the quantity that reduces by greater than 50 % the number of sRFC counted in a given field, as compared to controls.
RESULTS
Effects o f E L V inoculation on the levels o f S T F T h e levels o f S T F detected (at v a r i o u s time intervals after virus inoculation) in D B A / 2 mice i n o c u l a t e d either with heat-inactivated o r with v ar i o u s doses o f live F L V , are sh o w n in Fig. 1.
Serum thymic factor in Friend virus-infected mice
69
DBAI2 Mice 1/64
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"~ . . . .
- -
. O -J ~ . --
• '
. I ,'---D
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days after FLV administration FIG. 4. Same protocol as in Fig. 3. Symbols as in Fig. 1.
DISCUSSION The data presented in this paper clearly show that the administration of FLV to DBA/2 mice results in a pronounced decrease of the levels of STF and theta-positive splenic cells. These changes are detectable already on day 1 after virus inoculation, remain evident for about 4 weeks and tend to revert to normal values later on, even in animals with overt leukemia. Interestingly, the levels of thetaopositive cells of mice given undiluted FLV remained low even at 60 days after infection, whereas, with 10-1 and 10-2 FLV dilutions, their values bounced back to normality. One possible explanation is that the FLV-induced drastic decrease of STF levels, which the spleen theta-positive cells are very much dependent upon, has profoundly affected the recovery of these cells. Alternatively, one may envision a direct action of FLV on the splenic cells plasma membrane that may render them less sensitive to AZ. Maseio and Ceglowski [23] found a decreased sensitivity of mouse spleen cells, infected by FLV, to anti-theta serum. The two hypotheses are not mutually exclusive. The close parallelism of the kinetics of the two effects may indicate either an additive or a synergistic effect caused by lack of STF and FLV infection. In fact, the decrease of AZ spleen sensitivity caused by thymectomy [1] is neither as early nor as pronounced as that observed in our studies. The observed changes in STF levels and AZ sensitivity are dose-dependent, suggesting that they are related to virus replication. Additional evidence for this relationship is provided by the data observed in BALB/c mice given the same FLV preparations. BALB/c mice (Fv-1 b/b) are partially resistant to the replication of N-tropic strains of FLV. In keeping with data from the literature, our stocks of FLV showed a 3-1oglo reduction of titer when tested in BALB/c mice (data not shown). Under these conditions, both STF levels and AZ sensitivity were still reduced, in a dose-dependent fashion, by FLV inoculation but at a much lesser extent than in DBA/2 mice (Fv-ln/n). This indicates that a genotype partially restrictive for FLV replication is also restrictive for the observed modulation of the parameters under study.
72
ENRICO GARACIet al.
The mechanism by which FLV infection causes the observed drastic decrease of STF levels remains to be determined. A possible explanation could be provided by a direct effect of FLV on thymic epithelial ceils that actually secrete biologically active thymic products. We have, in fact, shown in various experimental conditions that the fall of STF levels is associated with profound alterations of these cells [14, 18, 19]. Preliminary observations, made with the electron microscope on day 2 and 16 after virus inoculation, do not appear to show widespread FLV replication in the epithelial cells. Still, negative data are never definitive because of the well known "sampling" error built in the ultrastructural studies and more work, therefore, is needed to resolve this issue. Another explanation may be as follows: the normal STF levels differ in DBA/2 and BALB/c mice. This suggests the possibility that mice with high constitutive STF levels may be more responsive to the "virus effect". The data described in this study do not allow us to draw an obvious correlation between the decrease of STF values and FLV leukemogenesis. As a matter of fact, overtly leukemic mice (more than 30 days after infection) did not have decreased STF levels and, moreover, had low levels of theta-positive spleen cells only occasionally (compare data in mice given undiluted and 10-1 FLV dilution). Yet, the peculiar kinetics of both effects (early and pronounced fall of STF and theta-positive cells in DBA/2 mice and to a lesser extent, in BALB/c mice) is suggestive of a possible pathogenetical relationship between these changes and the onset of FLV-induced leukemogenesis. One might ask the question whether the early fall in STF levels may play a role in the establishment of FLV infection and/or transformation of its target cells. In other words, the lack of STF soon after infection with FLV may cause an overall derangement of the immune system which may become less ready to fight and destroy the rapidly emerging leukemic cell population. The leukemic cells do possess new viral antigens and do also express hemopoietic histocompatibility antigens, that are not expressed in the corresponding normal hemopoietic cells [11, 12, 22, 29, 30]. The strength of the immune response against all these antigens may be very instrumental in determining the fate of the newly made leukemic cells. On the other hand, it is known that STF activity in man is decreased in several T-cell deficiencies and in advanced age [4]. In mice, STF activity decreases with age [5]; its administration, moreover, enhanced the ability of mice and rats to reject tumor grafts [20, 21, 34]. The conflicting evidence on the impairment of T-cell functions in FLV-infected mice [6, 8, 15, 16, 26, 2"1], may be accounted for by the fact that low STF titers do not necessarily induce an easily detectable defect of cell-mediated immunity. In fact, in adult thymectomized mice, the immediate fall of STF following removal of the gland is not accompanied by the simultaneous appearance of immunological changes thanks to the persistence of long-lived T-lymphocytes. Yet, some impairment of T-cell functions appears later [33]. Finally, it should be mentioned that the polycythemic strain of FLV, used in these studies, consists of a mixture of at least two viruses, i.e. the spleen focus-forming virus and the leukemia virus. The data presented here do not allow to attribute the observed changes to either one of the viruses. Studies in progress with the anemic strain of FLV (which lacks the focus-forming component, but induces an erythroleukemia indistinguishable from that induced by the polycythemic strain of FLV) will probably help to elucidate this point. Acknowledgements--The authors are indebted to Mr. G. Bagella for skilful technical assistance, and to Dr. V. Monaco for generous gifts of mice. This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CTB.77.01355.04 and Progetto Finalizzato Virus CT.77.00304.84).
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