Salmonella typhimurium was invariably isolated from our J strain murine leukosis. Immunization of D103 mice with either inactivated Salmonella typhimurium or the cell-free extract of leukosis inhibited the transplantation of leukosis. The adoptive immunization of D103 mice with spleen cells of Strong A mice immunized with either Salmonella or the cell-free extract of leukosis inhibited the transplantation of leukosis. The addition of either Salmonella or the cell-free extract of leukosis inhibited the migration of macrophages of leukosis spleen in tissue culture. Strong A mice is non-susceptible to J strain leukosis. However, inoculation of neonatal Strong A mice with the cell-free extract of leukosis produced a susceptibility to the transplantation of leukosis. These results suggest that both a filtrable agent and Salmonella typhimurium are present in cells of this leukosis and might be etiologically related to the leukosis.</p

Hamazaki, Yukio

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The immunological relationship between filtrable agent, Salmonella and murine leukosis

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Acta Medica OkayamaVolume 31, Issue 6 1977 Article 1DECEMBER 1977The immunological relationship betweenfiltrable agent, Salmonella and murine leukosisYukio Hamazaki∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.The immunological relationship betweenfiltrable agent, Salmonella and murineleukosis∗Yukio HamazakiAbstractSalmonella typhimurium was invariably isolated from our J strain murine leukosis. Immu-nization of D103 mice with either inactivated Salmonella typhimurium or the cell-free extract ofleukosis inhibited the transplantation of leukosis. The adoptive immunization of D103 mice withspleen cells of Strong A mice immunized with either Salmonella or the cell-free extract of leukosisinhibited the transplantation of leukosis. The addition of either Salmonella or the cell-free extractof leukosis inhibited the migration of macrophages of leukosis spleen in tissue culture. Strong Amice is non-susceptible to J strain leukosis. However, inoculation of neonatal Strong A mice withthe cell-free extract of leukosis produced a susceptibility to the transplantation of leukosis. Theseresults suggest that both a filtrable agent and Salmonella typhimurium are present in cells of thisleukosis and might be etiologically related to the leukosis.∗PMID: 147604 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 31, 343-349 (1977)THE IMMUNOLOGICAL RELATIONSHIP BETWEENFILTRABLE AGENT, SALMONELLA ANDMURINE LEUKOSISYukio HAMAZAKIDepartment of Pathology, Okayama Universit.y Medical School,Okayama 700, JapanReceived August 24, 1977Abstract. Salmonella typhimurium was invariably isolated from ourJ strain murine leukosis. Immunization of D103 mice with either in-activated Salmonella typhimurium or the cell-free extract of leukosisinhibited the transplantation of leukosis. The adoptive immunizationof D103 mice with spleen cells of Strong A mice immunized with eitherSalmonella or the cell-free extract of leukosis inhibited the transplan-tation of leukosis. The addition of either Salmonella or the cell-freeextract of leukosis inhibited the migration of macrophages of leukosisspleen in tissue culture. Strong A mice is non-susceptible to J strainleukosis. However, inoculation of neonatal Strong A mice with thecell-free extract of leukosis produced a susceptiblity to the transplan-tation of leukosis. These results suggest that both a filtrable agentand Salmonella typhimurium are present in cells of this leukosis andmight be etiologically related to the leukosis.The author has conducted long term studies on feeding young rats and miceon fresh human malignant tumor tissues obtained during surgery or autopsy.Since 1963 such experiments have been performed 85 times, and leukosis hasdeveloped 28 times (33 % ). In 18 of these strains, serial transplantations wereattempted. The cell-free extact induced leukosis when injected into mice within24 hours of birth but not in adult mice (1-4). Many inbred mice were susceptibleto this leukosis. The D103 mice were most susceptible, and the Strong A micewere not susceptible at all. This cell-free extract was rather stable and remainedactive for about 2 years if the spleen or liver tissue was immersed in 50% glycerinand kept at - 30°C. The leukosis was myelogenous and produced moderateenlargement of the spleen and liver, without any remarkable changes in peri-pheral blood. The term" leukosis" was used, as it more accurately describes theactual condition, than the term leukemia.It was of special interest that many Salmonella typhimurium bacilli were in-variable and purely isolated from the first passage from the spleen or liver of miceof the J strain of our leukosis.What implication does Salmonella have in this J strain leukosis? This paperreports on immunological studies on the relationships between the virus, Salmo-3431Hamazaki: The immunological relationship between filtrable agentProduced by The Berkeley Electronic Press, 1977344nella bacilli and this leukosis.Y. HAMAZAKIMATERIALS AND METHODSMice. D103 and Strong A mice obtained from the mouse colony of Oka-yama University Medical School were used.Leukosis. J strain murine leukosis transplanted in D103 mice was used.The history of this leukosis strain has been reported elsewhere (1-4).Antigens. Salmonella typhimurium isolated from the spleen and liver of D103mice afRicted with leukosis was used, and a suspension containing about 1,400bacilli in I cmm of physiological saline solution was prepared. This was in-activated by mixing with formalin to 0.5% at O°C for 24hr. The cell-freeleukosis extract was prepared by adding a five-fold volume of physiologicalsaline solution to the spleen and liver of leukosis mice; thoroughly homoge-nized at O°C; and passed through HAWP millipore filter to produce a cell- andbacteria-free extract. Control antigen was similarly prepared with the spleenand liver of normal D103 mice.Leukosis cell suspension for challange. Leukosis spleen and liver were mincedand suspended in physiological saline to 10%, and 0.03ml was intraperitoneallyinjected as a challenge.Immunization. The antigen solutions were mixed with an equivolume ofFreund's incomplete adjuvant (Difco). A half or one milliliter of antigen wasinjected into the extremities of mice at four different sites. The injections wererepeated 5-6 times at intervals of 5 days, and a month later the challenge sus-pension was injected.Adoptive immunization test. Salmonella bacilli antigen (O.lO.l5ml) or thecell-free extract of the leukosis spleen and liver (0.5-1.0ml) was intraperitone-ally injected into Strong A mice, which are non-susceptible to this leukosis.This was repeated three times at intervals of 5 days, and 5-7 days after the lastinjection, the mice were bled to death. Spleen lymphatic cells removed fromthese animals were intraperitoneally injected into recipient D103 mice. Thenumber of cells injected into each animal was about 5x 107• At 5 to 10 daysafter cell transplantation, the challenge leukosis cell suspension was intra-peritoneally injected.In the control group, a similar number of spleen cells of non-treated, normalStrong A mice were transplanted into D103 mice, and similarly challenged.Macrophage migration inhibition test. Leukosis spleen cells were intraperito-neally injected into D103 mice, and when the splenomegaly palpated, micewere bled to dea tho The spleen was removed, and tissue specimens of about2mm2 were obtained from the parenchyma. These tissue specimens were fixedto the bottom of small dishes about 4cm in diameter with plasma obtainedfrom Strong A mice, and maintained in Eagle's minimum essential mediumsupplemented with 20% calf serum. In the experimental group three antigenswere added to the culture medium: (a) suspension of Salmonella typhimuriumisolated from leukosis, (b) cell-free extract of the spleen and liver of leukosis,2Acta Medica Okayama, Vol. 31 [1977], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss6/1Immunological Studies on Murine Leukosis 345and (c) a mixture of these two antigens. The optimum antigen amount was0.2ml per 5ml of culture medium. After culture for 24 hr at 37°C in a 5% CO2incubator, the culture medium was removed, floating cells (mostly lymphocytesand leukocytes) were washed off, and cells fixed to the glass surface (mostlymacrophages) were rapidly dried by a fan, fixed and stained by Giemsa stain.The migration distance was measured under a microscope with an ocularmicrometer. Measurements were performed in six spleen specimens, and theaverage migration distance was computed.Immunological tolerance test. Newborn Strong A mice non-susceptible toleukosis were used as test animals. As pretreatment 0.1-0.15 ml of the cell-freeleukosis extract was intraperitoneally injected into newborns. At 35 to 40 daysafter pretreatment, mice were given intraperitoneal injections of the leukosiscell suspension.RESULTSImmunization with Salmonella and celljree extractD I03 mice immunized with Salmonella from leukosis and challenged withthe leukosis cell suspension showed an average survival period of 49 days whilecontrol animals showed a survival period of 16 days (Table 1).TABLE 1. IMMUNIZATION WITH Salmonella AND CELL-FREE EXTRACTDays from leukosis cellNo. of mice" t~nsp~antationto!~~osis deathAntigen used forimmunizationSalmonella from leukosis/)NoneCell-free extract of leukosis spleen and liverCell-free extract of normal spleen and liver6566Range20- 669- 1239-1125- 11Mean4916618a D103 mice immunized with antigen and challenged a month later with leukosis cellsuspension.Inactivated with formalin.When the cell-free extract of leukosis was injected and challenged with thecell suspension of leukosis, the average survival period was 61 days, and it was8 days in the control group (Table I).Adoptive immunityA general principle in adoptive immunity is that live cells be used as antigen(5). Salmonella may thus be antigen in adoptive immunity, but viruses are notcells in this sense. A rule in virus use as antigen in adoptive immunity is that thesensitive animals are first infected with the virus, and the virus-infected cells areused as antigen in adoptive immunity (6). However, our murine leukoses wereinvariably infected with Salmonella, and virus-infected cells, void of Salmonella3Hamazaki: The immunological relationship between filtrable agentProduced by The Berkeley Electronic Press, 1977346 Y. HAMAZAKIcannot be collected (7, 8). Therefore, the cell-free leukosis extract was used asthe antigen.Antigen from live Salmonella cultured on agar slopes for 24 hr produced anextended life span in the experimental group (Table 2, Experiment I).On the other hand, when inactivated Salmonella was used as antigen, nodifference in survival was observed between the experimental and control mice(Table 2, Experiment II and III).When the cell-free extract of leukosis was used as antigen, the life span ofmice of the experimental group was significantly extended in comparison withthat of control animals (Table 2, Experiment IV). When the cell-free extractwas heated to 56°C for 30 min, no effect was observed (Table 2, Experiment V).Macrophage migration inhibitionIn specimens cultured in the absence of antigens, macrophage migration wasvery clearly observed (Fig.la). Cells lying parallel extended widely and displacedthe substrate (coagulated plasma) on the glass surface to form a coating in theperiphery. On the contrary, in specimens cultured in the presence of antigens,migrating cells aggregated irregularly about the tissues, and the migration dis-tance was short (Fig. 1b).The migration index was calculated according to the following formula, inwhich MI means the migratory index, and MD l and MD 2 are the mean valuesof the migration distances in the control and antigen-added specimens respec-tively.MI= -- ¥Q~-- X 100MD1When Salmonella bacilli were used as antigen, MI was 33%; when the cell-free extract of leukosis was used as the antigen, MI was 32%; and when amixture of these two antigens was used as the antigen, MI was 21 % (9).Immunological toleranceStrain Strong A is non-susceptible to our leukosis. Of 39 Strong A mice in-oculated at birth with the cell-free leukosis extract, none developed leukosis(Table 3, Control A). Another group of 30 Strong A mice receiving no treatmentat birth and inoculated with the leukosis cell suspension at 35-49 days of age alsodid not develop leukosis (Table 3, Control B). However, in 39 mice inoculatedwith the cell-free leukosis extract at birth and again inoculated with the leukosiscell suspension 35-49 days later, 18 (46%) developed leukosis (Table 3, Experi-mental group). In comparison with leukosis in DIG3 mice, the liver and spleenenlargement in leukosis-developing Strong A mice was not remarkable, and thehistological changes were mild. This was especially clear in mice developingleukosis after a short latent period of several weeks.4Acta Medica Okayama, Vol. 31 [1977], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss6/1TABLE 3. INDUCTION OF IMMUNOLOGICAL TOLERANCE BY CELL-FREE EXTRACTPretreatment Challenge by No. of miceDays from leukosis cellGroup No. of mice" developing transplantation to leukosis deathat birthb leukosis" leukosis (%) Range Mean..-------Experimental 39 Done Done 18 (46) 32-311 160Control A 20 Done Not done 0(0)Control B 20 Not done Done 0(0)No. ofexperimentIIIIIIVVTABLE 2. ADOPTIVE IMMUNITYAntigen used for No. of No. of mice Days from leukosis cellGroup immunization of recipient developing transplantation to leukosis deathdonors mice leukosis Range Mean~~--_._---~-_..-Experimental a Live Salmonel/a b 5 5 42-115 63Control" 5 5 5- 10 7Experimental" Formalin-treated Salmonellab 5 5 6- 16 10Contro( 5 5 7- 16 12Experimental" Heated Salmonel/a b• d 5 5 5- 13 9Control" 5 5 6- 10 8Experimental" Cell-free extract of leukosisb 5 5 12-- 27 17Control" 5 5 6- 12 9Experimental" Heated cell-free extract of 5 5 4- 10 7Control" leukosisb• (j 5 5 4- 14 7a D103 mice received spleen cells of Strong A mice immunized with antigens, and 5-10 days later, received a challenge ofleukosis cell suspension.Isolated or made from the spleen and liver of leukosis mice.D103 mice received spleen cells of normal Strong A mice and were treated in the same way as experimental mice.d Heat-treatment was conducted at 56°C for 30 min.a Strong A mice non-susceptible to this leukosis were used.Intraperitoneal inoculation of cell-free leukosis extract within 24 hours after birth.c Intraperitoneal inoculation of leukosis cell suspension at 35-49 days of age.3'sC::lo0'(Ill0'e:.CJJ2Q.~.o::l~C...~.t""'(11c:>;'o~.'"c.>:l~-I5Hamazaki: The immunological relationship between filtrable agentProduced by The Berkeley Electronic Press, 1977348 Y. HAMAZAKIFig. I. Inhibition of macrophage migration. (al, Control in which Salmo7ellatyphinurill n was not added to the culture-medium. May-Giemsa. ~< 40. (bl, Spleentissue fragment of J stain murine leukosis cultured in the presence of Sa/mo lellat),phi.'Tluriull isolated from the same strain of leukosis. May-Giemsa. ,< 40.DISCUSSIONThe most remarkable characteristic of our J strain leukosis was the invari-able isolation of Salmonella typhimurium from leukosis tissues. In leukosis inducedby inoculating cell-free extract into newborn D103 mice, a large amount ofSalmonella bacilli was invariably isolated from the spleen and liver in the firstpassage. This suggests that for the development of J strain leukosis, the syner-getic action of specific Salmonella bacilli and a filtrable agent, probably a virus, isnecessary.In immunization test with DIG3 mice, treatment with either Salmonella orthe cell-free extract of J strain leukosis inhibited the transplantation of leukosis.In adoptive immunity studies, transplantation of spleen cells of Strong A miceimmunized with either live Salmonella or the cell-free extract significantly in-hibited leukosis cells in mice. In macrophage migration inhibition studies, theaddition of Salmonella bacilli or the cell-free extract remarkably inhibited macro-phage migration and when these two factors were simultaneously added to theculture medium, a more remarkable inhibitory effect was observed. These re-sults suggests that both a filtrable agent and Salmonella typhimurium exist in andare etiologically related in our J strain leukosis. An immunologic tolerance toleukosis transplantation was successfully induced in Strong A mice by inoculatingthe cell-free extract of leukosis at birth. This also suggests the presence of afiltrable agent in cells of our J strain leukosis.The existence of leukemia virus in leukemia cells is well known in mice(10). Salmonella bacilli unlike common bacilli have the property of proliferatingas L type bacteria in cells they occupy (facultative intracellular parasitic bacteria).For this reason they might make contact with a filtrable agent within the cell andby synergetic action with it, disturb the nucleic acid metabolism of host cells (11,12).Acknowledgment. The author wishes to express his appreciation to Prof. Y. Yabe, Prof.Y. Kanemasa and Dr. K. Orita, Lecturer, for their kind help and discussions.6Acta Medica Okayama, Vol. 31 [1977], Iss. 6, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol31/iss6/1Immunological Studies on Murine LeukosisREFERENCES349I. Hamazaki, Y.: Experimental induction of lymphatic leukemia in rats following inges-tion of human neoplasms. Z. Krebiforsch. 67, 233-246, 1965.2. Hamazaki, Y.: Experimental induction of leukosis following ingestion of human neo-plasms. j. Karyo,pathol. 11, 1-4, 1966.3. Hamazaki, Y. and Matsuura, Y.: Experimental induction of a new type of murineleukosis following ingestion of human neoplasms. Arch. GeschwulstJorsch. 39, 107-120,1972.4. Hamazaki, Y., Murao, T. and Murao, T.: Experimental studies by tissue culture of anew murine leukosis virus. Arch. GeschwulstJorsch. 39, 8-23, 1972.5. Billingham, R. E., Brent, L. and Medawar, P. B.: Quantitative studies on tissue trans-plantation immunity. II. The origin, strength and duration of actively and adoptivelyacquired immunity. Proc. R. Soc., B. 143, 58-80, 1954.6. Aoki, T., Old, L. j. and Boyse, E. A..: Specific antibody in leukemic mice. Saishin-Tgaku.21, 2343-2351, 1966 (in japanese).7. Kawakami, M., Ishibashi, H., Mitsuhashi, S., Sakaino, K. and Fukai, K.: Experimentalsalmonellosis. Unstable L forms in liver of infected mice. ]pn.,T. Microbial. 14, 143-153,1970.8. Kawakami, M. and Mihashi, S.: Infection and cellular antibody. In Cellular Immunity,ed. T. Kuroyanagi, Y. Otaka and T. Matuhashi, Igakushoin, Tokyo, pp. 112, 117, 1969(in japanese).9. Yoshida, Y.: Detection of cellular antigen. In Cellular Immunit)" ed. T. Kuroyanagi, Y.Otaka and T. Matuhashi. Igakushoin, Tokyo, p.270, 1972 (in japanese).10. Aoki, T., Boyse, E. A. and Old, L. J.: Occurrence of natural antibody to G(gross) leuke-mia antigen in mice. Cancer Res. 26, 1415-1419, 1966.11. Ames, B. N., Smis, P. and Grover, P. L.: Epoxides of carcinogenic polycyclic hydrocar-bons are frameshift mutagens. Science 176, 47-49, 1972.12. Smith, T. T. and Bridges, R. A.: Immunological unresponsiveness in rabbits producedby neonatal injection of definite antigens. ]. Exp. Med. 108, 227-250, 1958.7Hamazaki: The immunological relationship between filtrable agentProduced by The Berkeley Electronic Press, 1977

A..: Specific antibody in leukemic mice.

and Murao,T.: Experimental studies by tissue culture of a new murine leukosis virus.

Detection of cellular antigen. In Cellular Immunit)&quot;

Epoxides of carcinogenic polycyclic hydrocarbons are frameshift mutagens.

Experimental induction of a new type of murine leukosis following ingestion of human neoplasms.

Experimental induction of leukosis following ingestion of human neoplasms.

Experimental salmonellosis. Unstable L forms in liver of infected mice.

Immunological unresponsiveness in rabbits produced by neonatal injection of definite antigens.

Infection and cellular antibody.

Occurrence of natural antibody to G(gross) leukemia antigen in mice.

Quantitative studies on tissue transplantation immunity. II. The origin, strength and duration of actively and adoptively acquired immunity.

Y.: Experimental induction of lymphatic leukemia in rats following ingestion of human neoplasms.

The immunological relationship between filtrable agent, Salmonella and murine leukosis

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