Isolation and activity of a thymocyte-transforming factor from the mouse submaxillary gland

Experimental

ISOLATION

AND

ACTIVITY

Cell Research 57 (1969) 95-103

OF A THYMOCYTE-TRANSFORMING

FROM THE MOUSE SUBMAXILLARY M. A. NAUGHTON,

FACTOR

GLAND

J. KOCH, H. HOFFMAN, V. BENDER, H. HAGOPIAN and E. HAMILTON

CSIRO, Division of Animal Genetics, Epping, N.S. W. 2121, Australia

SUMMARY A protein has been isolated, in electrophoretically homogeneous form, from mouse submaxillary gland, which induces, both in vitro and in vivo, transformation of thymic small lymphocytes into cells of the plasma series, without cell division, and with considerable increase in cytoplasmic volume, and RNA content. This transformation is accompanied at higher concentration of the protein, by agglutination and lysis.

The injection of crude extracts of mouse submaxillary glands into new-born mice was shown by Levi-Montalcini & Cohen [8] to induce runting. This observation was confirmed by Bueker & Schenkein [2] who also observed atrophy of the thymus and other lymphoid organs and their depopulation of lymphocytes. They reported that as soon as regular administration of the extract was stopped, the animals recovered from the runting condition and grew normally. Takeda et al. [13] injected crude extracts of mouse submaxillary glands into adult mice and observed lymphopenia and atrophy of lymphoid organs; this atrophy consisted primarily in reduction of the small lymphocyte population in these organs. They showed the effect could be obtained in adrenalectomised animals and so was not mediated by corticosteroids. Takeda & Grollman [14] showed that the greater part of the substance in the submaxillary gland, active in inducing atrophy of thymus, could be located in a specific protein fraction of prepared extracts. Small lymphocytes from newborn mouse thymus, when cultured on slicesof electrophoretic gels prepared from mouse submaxillary gland, were shown by Hoffman et al. [7] to be lysed in the region of one specific band (R, 0.51). This

was later confirmed by Liuzzi & Angeletti [lo] using partially purified extracts. In a further study Hoffman & MacDougall demonstrated that this region of the gel lysed small lymphocytes from the thymus and mesenteric node, but failed to lyse morphologically indistinguishable cells from either quiescent or antigenactivated femoral lymph node [6]. A similar lytic band was demonstrated in electrophoretic gels prepared from a homogenate of bovine parotid gland. This paper is concerned with the isolation of the substance in the mouse submaxillary gland responsible for lysis of thymic lymphocytes and with the characterisation of its biological activity. It describes the preparation of crude extracts from the mouse submaxillary gland, the purification of the active substance in these extracts, and in vivo and in vitro tests of the crude extract and the purified substance. MATERIALS AND METHODS Preparation of crude submaxillary gland extracts Submaxillary glands from 25 male mice were homogenised in 50 ml of 0.25 M sucrose solution for 2 min in a high speed Virtis homogeniser. The mixture was centriExptl Cell Res 57

96 M. A. Naughton et al. fuged for 15 min at 1700 g; the supernatant was collected, and the residue, after being resuspended in the same medium, was centrifuged again. The combined supernatants were then centrifuged at 10,000 g for 30 min, the residue was resuspended in 0.25 M sucrose and the treatment repeated. The washed sediment was then resuspended in distilled water, at a concentration of about 1 mg/ml and ultrasonically disrupted, providing the crude submaxillary extract.

Preparation and purification of protein For the initial fractionation of submaxillary gland proteins the method of Varon et al. [15, 161 was used, all procedures being carried out at 4°C. Submaxillary glands of 100 male mice (w/w 15 g) were homogenized in 300 ml of distilled water. The homogenate was centrifuged at 50,000 g for 1 h in a Spinco L centrifuge. The pink supernatant (260 ml) was freeze-dried and dissolved in 18 ml of 0.05 M Tris/HCl buffer at pH 7.4; 9 ml of the solution was applied to a G 100 Sephadex column (100 x 2.5 cm) equilibrated with 0.05 M Tris/HCl buffer at pH 7.4. The effluent from the column was monitored at 280 rnp with a Beckman DB spectrophotometer. Samples of the fractions were freeze-dried and dissolved in Hanks’ solution to produce a final concentration of approx. 0.25 to 0.5 OD units/ml (measured at 280 mp) and assayed as described below. Following assay, the fractions corresponding to peak 2 in text-fig. 1 were combined, the pH adjusted to 9.5 with NaOH solution, and the protein adsorbed onto a column of A 50 DEAE Sephadex equilibrated with 0.05 M Tris/HCl buffer 9.5 nH. The adsorbed nrotein was eluted with a linear gradient of 900 ml of 0105 M Tris/ HCI against 900 ml of 0.05 M Tris/HCl with 0.05 M NaCl at a flow rate of 0.8 ml/min.

Assay of extract on tissue culture Thymus from newborn to three-day-old Quackenbush mice was excised and finely divided in ice-cold Hanks’ solution. Fragments were explanted onto thin discs of 7.5 % acrylamide gel, mounted within teflon rings on microscope slides. The gel discs were prepared from large sheets of gel, with the aid of a sterile cork-borer, then soaked exhaustively in a number of changes of Hanks’ solution containing 5 ,ug/ml of Terramycin. Medium was then noured over the explants and the cells sealed by waxing on thin coverslips. The media used were fowl plasma, prepared with minimal EDTA, with the Ca2+ concentration restored after centrifugation, and bovine thrombin in minimal quantity to clot the plasma; sheep and mouse plasma, prepared similarly; fowl or sheep serum. Experimental cultures had submaxillary gland extract or fractions from the column separations added to them; these additions were made up of 1 vol of material diluted in 9 vol of culture medium, and were added to a set of cultures; further cultures received serial dilutions, each l/&h the concentration of the previous one. Cultures were examined live, usually after 48 h incubation, by phase contrast microscopy, using a x 40 Zeiss-Oberkochen water-immersion phase objective, n.a. 0.75. Similar cultures were fixed in Formol-Muller’s solution, imbedded in glycol-methacrylate [13], sectioned and stained with pyronin-methyl green. Other cultures were fixed in glutaraldehyde, followed by osmium tetroxide, sectioned in Epon, and examined in the electron microscope. Exptl

Cell Res 57

Testing extracts on mice Fractions from the various stages of purification were injected into both newborn and 8-12-week-old mice for varying periods, after which the animals were killed and their tissue fixed, imbedded and stained for microscopic examination, using the methods already detailed for tissue cultures. When newborn mice were injected, half of each litter was injected with submaxillary extract, starting at 0.02 ml/day and rising with increasing weight of mouse, while the other half of the litter received Hanks’ solution only. Altogether 11 litters totalling 94 animals were treatedmost were injected for 3-4 weeks, then killed; one group was injected for 9 weeks. Adult Quackenbush mice were injected with extracts dissolved in Hanks’ solution to an OD of 0.5 units/ml; 17 received the extracts and 17 Hanks’ solution only. The dose was 0.1 ml of solution injected subcutaneously daily for from 4 to 14 days. Colchicine was used to examine any modification of the mitotic pattern in the thymus of injected animals. The dose was 3 mg/kg intraperitoneally, the animals being examined 6 h after injection.

RESULTS Control The surface of gels made up with control medium became populated by migrating cells within hours of explantation of tissue fragments. More than 95 % of the cells migrating over the gel surface were small lymphocytes, such as those illustrated in fig. 1, the remainder were mostly blast cells, usually with very small but variable numbers of plasma cells. Crude extracts Lymphocytes from tissue fragments that had been explanted onto gels containing high concentrations of crude extracts of submaxillary gland (dilution 1O-1-2)showed considerable lysis and agglutination. The cells assembledin chains or clumps, then lysed, coming to resemble the “ghosts” of lysed red cells. As the concentration was lowered by diluting to 1O-3 and beyond, fewer cells were lysed, agglutination was no longer observed, and many of the surviving cells underwent a characteristic transformation in which the nucleus remained the same size, but developed clear areas of low density with a prominent nucleolus and became eccentrically placed in a dense, foamy cytoplasm of considerably increased volume. A vacuolar Golgi system becameconspicuous in a site next to the nucleus.

1 I

1.0

3.0

Thymocyte-transforming

factor from mouse submaxillary gland

91

2.0

1.0

i

d

Text-fig. 1. Fractionation of the protein extracted from the mouse submaxillary gland on Sephadex G 100 (6.5 ml/fraction). The diagrams in the upper part of the figure show the acrylamide gel ionogram patterns of the proteins across the various peaks.

Purified extracts

The profile in text-fig. 1 shows the optical density at 280 rnp of successivefractions of submaxillary gland extract after separation on a GlOO Sephadex column, together with the acrylamide gel ionograms of 100,ul samples of the indicated fractions. When the various major peaks shown in text-fig. 1 were assayedwith thymic lymphocytes, transforming and lytic activity was found only in peak 2. The fractions in peak 2 were then further fractionated on an A 50 DEAE Sephadex column, yielding a separation illustrated in textfig. 2, which shows the profile of OD at 280 m,u of the fractions separated on the A 50 DEAE Sephadex column together with the acrylamide gel ionogram diagrams of the various fractions. High activity was found in the large peak [4] of text-fig. 2 in successivepreparations carried out by this method; fractions in this peak were used to prepare the purified factor which was freezedried and transferred to physiological solutions via Sephadex G25. The gel ionogram reveals that this fraction is electrophoretically homogeneous. 7- 691805

This preparation proved to have high activity in vitro-in highest concentration it induced considerable lysis and agglutination, whilst dilution to 1O-5and beyond resulted in diminution of agglutination and lysis, with concomitant increasein the proportion of cells undergoing the very characteristic transformation mentioned earlier. Sometimes up to 80% of the lymphocytes in the culture were transformed, showing all stages from modified small lymphocytes to the extreme stages of transformation which are indistinguishable from the typical plasma cell. Such an area is illustrated in fig. 2, in which almost every cell deviates markedly from the control small lymphocytes illustrated in fig. 1. As the preparation was further diluted, the cultures gradually cameto resemblethe control ones more closely. With potent fractions this occurred at 1O-6,or beyond. When sheep or mouse instead of fowl serum or plasma was used cells appeared to transform in much the same fashion as in fowl medium; many of the transformed cells acquired the asymmetric nucleus, large nucleolus, bulging, dense Exptl Cell Res 57

98 Al. A. Naughton et al. 24

1.5

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0.5

i n 1'0

1'5

2'0

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4'5

5'0

5'5

do

6'5

f0

Text-fig. 2. Fractionation of the proteins from peak 2 from text-fig. 1, on DEAE Sephadex A 50 using a gradient pH 9.5,0.05 M Tris-HCI to pH 9.SO.05 M Tris-HCI and 0.5 M NaCl, 10.5 ml fractions. Diagrams from the acrylamide gel ionograms from the fractions across the various peaks are shown in the upper portion of the figure. Fractions 56 to 60 of the main peak were used to prepare the thymotropic factor.

foamy cytoplasm, and enlarged Golgi zone; some cells deviated somewhat, becoming enlarged without the nuclear displacement, as seenin figs 7, 8. On the other hand, marked differences were observed in the manner of lysis; in mammalian medium “ghosts” were not seen; instead, the cells became severely shrunken and pycnotic, as

illustrated in figs 6a, 7, 8 (lines), and naked pycnotic nuclei were often encountered. These pycnotic, shrunken lymphocytes closely resembled those seen in the circulating blood of mice injected with active submaxillary fractions, as will be described later. The most characteristic forms of transformed

Figs l-8 photographed live, phase contrast, magnification x 800. Figs l-5 (c) incubated in fowl plasma, figs 6-8 incubated in the sheep plasma. Fig. 1. (a) Two fields showing normal small lymphocytes in control cultures of newborn mouse thymus, incubated 48 h. The cells are all similar, quiescent small lymphocytes. Fig. 2. Portion of a culture of newborn mouse thymus incubated 48 h with medium containing 10 % by volume of a crude submaxillary gland preparation. Most of the cells have undergone considerable transformation, by comparison with control specimens fig. 1, 1 (a). Arrows indicate cells which most closely resemble mature plasma cells. In these, nuclear density is considerably reduced in some regions, nucleolus has become prominent, cytoplasm has increased in volume, density and become foamy. Fig. 3. (a) A group of cells incubated with an active thymotropic preparation. Considerable agglutination has occurred, most of the agglutinated cells are lysed, but one surviving cell shows the characteristic plasma cell appearance (arrow). (b) A small group of agglutinated cells, lysing, while one cell in the group is transformed (arrow). Fig. 4. (a) One transformed cell with dense cytoplasm and prominent nucleolus (arrow) lies separated from a chain of agglutinated, lysed ones. Note the vaccuole near the nucleus. (6) In the centre of the photograph is an agglutinated mass of cells, of which some appear lysed, others are intact. Alongside these are two enlarged, transforming cells (arrows) of characteristic form with well developed vaccuolar system. Cultures incubated with 10-a purified thymotropic protein. Fig. 5. (a, b) These two figures show similar cells with small eccentric nuclei, prominent nucleoli, and dense, foamy cytoplasm. (c) A variety of transformed cells are shown in this figure, indicated by arrows: in particular, the ~11 at p, closely resembles the cell seen in circulating blood in fig. 12 (b). Fig. 6. Normal newborn mouse thymus lymphocytes, incubated in sheep plasma, control specimen for figs 6 (a)-8. Fig. 6. (a) Considerably enlarged transforming lymphocytes, in sheep plasma containing lO+’ dilution of protein solution obtained from peak 4 (text-fig. 2); (b) A very typical mature plasma cell, with eccentric nucleus, dense cytoplasm and juxtanuclear vacuole. From the same culture as fig. 6 (a). Enptl Cell Res 57

Thymocyte-trcnsforming

factor from mouse submaxillary gland

99

100 M. A. Naughton et al. cells have extreme nuclear heterogeneity, an enlarged nucleolus, foamy dense cytoplasm eccentrically arranged and of considerably increased volume, and well developedjuxtanuclear Golgi vacuoles; this is well illustrated in various figures (e.g. 3a, b; 5a, b; 6c) and is highly suggestive of the plasma cells seen in fixed tissues. In stained sections of fixed cultures, these transformed cells were seen to be, in fact, typical plasma cells with dense pyroninophilic cytoplasm. Electron microscopic studies generally supported this view: the juxtanuclear vacuole system was seento be a highly elaborated Golgi system, the cytoplasm was packed with dense massesof ribosomes, and endoplasmic reticulum components were distended and contained dense amorphous material. These appearances are all suggestiveof a plasma cell transformation of the cultured lymphocytes. Never in any of these experiments in vitro has it been possible to separate a fraction causing lysis and agglutination from one causing transformation-even when using electrophoretically homogeneous preparations. Separation of lysis and agglutination from transformation occurs when serial dilution is practised; further, the dilution pattern-lysis and agglutination in higher concentrations, and transformation in lower concentrations-was constant. These observations lead to the conclusion that a single substance is responsible for lysis and agglutina-

tion and transformation but not necessarily that these effectsare consequencesof a single activity. Treatment of 48 h old cultures, which had been incubated with active protein preparation and in which the transformation of lymphocytes was proceeding actively, with colchicine at the rate of 0.4 pug/mlfailed to reveal any mitotic figures, nor did incubation of cultures for the full 48 h period with this concentration of colchicine inhibit transformation. Injection of active protein into mice Adults: Daily injection of the active protein into adult mice at 0.05 OD units per mouse, produced detectable changes in the blood picture after 24 h. The total number of blood cells fluctuated little but there was considerable change-shrinkage and pycnosis-in many of the small lymphocytes, which came to resemble those grown in mammalian culture medium with active protein, described earlier in this paper. After 5 days of injection of protein, half of the lymphocytes were pycnotic, while more than 20 % of the remainder had enlarged and undergone a transformation similar to that seen in cultures incubated with the active protein. Transformed lymphocytes, markedly reminiscent of plasma cells, are shown in fig. 12a, b and may be contrasted with the normal lymphocytes (fig. 11a, b). Note the increased cytoplasmic volume of transformed cells, while fig. 12a clearly shows

Fig. 7. Portion of a culture incubated in 1O-2dilution of solution of the peak 4 protein, in sheep plasma. Large trans-

forming cells are shown (arrows) together with shrunken, pycnotic cells (lines) and some normal lymphocytes. Fig, 8. Some extremely large transformed cells with small, modified nuclei; one with very prominent nucleolus is shown

at x. In these cells the cytoplasm is extremely dense. Lines point to shrunken lymphocytes-some are little more than moribund, naked nuclei. Fig. 9. A section through the cortex of the thymus of a control 8 week old mouse injected daily for four days with Hanks’ solution only (0.1 ml/day)-the surface of the thymic lobe appears at top right. A small number of mature plasma cells may be detected (arrows) but the cellular population is largely made up of typical small lymphocytes. Fixed Formol-Muller, imbedded and sectioned in nlvcol methacrvlate, stained uvronin-methyl green. x 600. Fig. 10. This photograph represents a similar section through the thymic cortex of an 8 week aid mouse injected daily for 4 days with 0.1 ml of thymotropic factor 0.25 OD units/ml in Hanks’ solution. The surface of the thymus is shown in the upper left. It may be readily observed that the cell. population is markedly different from that-of the control (fig. 9). The small lymphocytes are largely gone, and in their place is a rather homogeneous population of larger cells with larger, pale nuclei and narrow rims pf pyroninophilic cytoplasm, reminiscent of plasmablasts. Technical details as for fig. 9. x 600. Fig. Il. (a, b) Normal medium lymphocytes from blood smear of control mouse, stained Giemsa, x 600. Fig. 12. (a, b) Plasma-type cells seen in smear of blood from a mouse injected with thymotropic protein 0.1 ml/day of a solution containing 0.25 OD units/ml for 6 days. These cells clearly show considerable increase in cytoplasmic volume, and the cell in fig. 12 (a) is very basophilic with a well developed juxtanuclear Golgi zone. Giemsa, x 600. These cells in smears cannot be compared directly for size with cells in sections. Smearing flattens and therefore apparently enlarges free cells, while imbedding may even cause shrinkage. Exptl Cell Res 57

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Exprl Cell Res 57

102 M. A. Naxghton et al. plasma type cells or lyses them, in vivo and in vitro. Since this dual effect is selective to some degree-lysis does not occur in cells from regional nodes-we have renamed this factor “thymotropic factor”. A variety of agents have been described which transform small lymphocytes into lymphoblasts or plasmablasts in vitro. Among these are phytohaemagglutinin (PHA), pokeweed mitogen [5] and bacterial antigensin various forms (for bibliography, see [9]). Each of these agents produces a different and distinctive cellular response: PHA agglutinates the lymphocytes, which proceed to transform into lymphoblasts; these then divide successively.Eventually PHA-transformed cells come to resemble the earliest stagesin the plasma cell series, but can never be induced to develop into true plasma cells [3]. Pokeweed mitogen induces some cells to divide; others develop directly in the plasma cell direction [4] even if mitosis is prevented [12]. Pokeweed also appears to act effectively in vivo: African children who ate pokeberry developed a syndrome in which numerous plasma cells appeared in the circulating blood [I]. Thesevarious agents have features in common with the thymotropic factor described here. Thus PHA and thymotropic factor agglutinate lymphocytes, both pokeweed and the thymotropic Animals injected from birth, for four weeks factor can transform small lymphocytes directly Similar extensive changes occurred in mice in- to plasma-like cells. However, this direct transjected daily from birth: the animals underwent formation of a small lymphocyte to a plasma characteristic runting. Although the thymus did cell, in the absence of nuclear enlargement or not reveal the same homogeneous plasmablast mitosis, accompanied by considerable increase in population, thymus of injected animals differed cytoplasmic volume and ribosome content, is from thymus of controls both in the increased very different from the effect of PHA or bacterial prevalence of plasma cells, and reduction in num- antigens. The finding of plasma cells in the circulating bers of ambient small lymphocytes-further, in treated thymus there were found numerous pyc- blood, and the considerable changes wrought in thymus and lymphoid organs by administration notic and moribund small lymphocytes. of this protein all suggestthat, since it has been isolated from a secretory gland, it may be perDISCUSSION forming some physiological role in the animal. The evidence presented above supports the con- The presence of rather immature cells of the clusion that the electrophoretically homogeneous plasma series in large numbers in the thymic protein isolated from the mouse submaxillary cortex, while more mature forms are found in gland transforms thymic small lymphocytes into the blood and lymphoid organs of injected anithe increased cytoplasmic basophilia, and the highly developed juxtanuclear Golgi region. Characteristic changes also took place in the thymus, and to some extent other lymphoid organs. The thymic cortex of adult control mice, injected with Hanks’ solution only, was predominantly composed of small lymphocytes (fig. 9). The epithelial, reticular and mature plasma cell components were on the whole inconspicuous. In contrast, the thymic cortex of animals injected for 4 days or more with active protein fraction was largely devoid of small lymphocytes; in their place was a relatively homogeneous population of larger cells eachwith a large pale nucleus, and a narrow rim of highly pyroninophilic cytoplasm. These cells were reminiscent of early plasmablasts (fig. 10). When animals were injected daily for 14 days, somewhat more mature plasmablasts were seenin the thymic cortex, but typical plasma cells were never observed in numbers in this site. On the other hand, red pulp of spleenand diffuse tissue of mesenteric node were heavily laden with typical mature plasma cells. Animals which had been injected for 6 days with the active protein did not reveal any increase in mitotic rate in the thymic cortex, as indicated by treatment with colchicine 3 mg/kg, and examination 4-6 h later.

Exptl Cell Res 57

Thymocyte-transforming

mals, supports the suggestion that after undergoing the first stages of transformation in the thymic cortex these cells migrate in the blood to other lymphoid sites, such as splenic pulp, and mesenteric node medulla. The lympholytic effects of this agent deserve further attention. The action in vivo is quite characteristic-there is no generalised destruction of the stroma or stem cells in lymphoid organs. Only mature small lymphocytes are lysed in mice injected regularly from birth, in contrast to the overall destructive action of corticosteroids. This highly elective destruction of small lymphocytes also helps to explain the ready reversibility of runting, described by Beuker & Schenkein [2] since the stem cells are unaffected and can rapidly regenerate a new generation of small lymphocytes. The final product of our fractionation was electrophoretically homogeneous, and as purification proceeded, activity rose in relation to concentration, so that the purified product was more active than the input, when diluted to the same volume of solution; thus we recovered the bulk of the activity in one homogeneous protein. Further it is possible that contaminating proteins in the crude preparation reduced its biological activity. Since we have been unable to separate lytic and transforming activity in our fractionation procedures, we are obliged to postulate that these different actions are the properties of a single molecular species. This agent bears no relationship to the high molecular

factor from mouse submaxillary gland

103

weight Nerve Growth Factor (NGF) described by Varon et al. [15, 161;this protein occurred in peak 1 (text-fig. 1) on Sephadex G 100, where no thymotropic activity occurs, while the thymotropic factor was concentrated in peak 2, which had no NGF activity. We wish to thank Mrs C. Cox, Mrs M. Blamires and Miss P. Perry for their technical assistance in this project.

REFERENCES 1. Barker, B E, Lutzner, M A, Farnes, P & Lamarche, P H, Clin res 15 (1967) 271. 2. Bueker, E D & Schenkein, L, Ann N Y acad sci 118 (1964) 183. 3. Chapman, J A, Elves, M W & Gough, J, J cell sci 2 (1967) 359. 4. Chessin, L N, Borjesson, J, Welsh, P D, Douglas, S D & Cooper, H L, J exptl med 124 (1966) 873. 5. Farnes, P, Barton, B E, Brownhill, L E & Fanger, H, Lancet 2 (1964) 1100. 6. Hoffman, H & MacDougall, J, Exptl cell res 51 (1968) 485. I. Hoffman, H, Naughton, M A, Hamilton, E A & MacDougall, J, Nature 214 (1967) 703. 8. Levi-Montalcini, R & Cohen, S, Ann N Y acad sci 85 (1960) 324. 9. Ling, N R, Lymphocyte stimulation. North Holland, Amsterdam (1968). 10. Liuzzi, A & Angeletti, P U, Experientia 24 (1968) 1034. 11. Ruddell, C L, Stain technol42 (1967) 119. 17 I-. Schwartz, M R, Anat ret 160 (1968) 47. 13. Takeda, T, Yamasaki, Y, Yamabe, H, Suzuki, Y, Haebara, H, Irinos, T & Grollman, A, Proc sot exntl biol med 126 (1967) 212. 14. Ta&da, T & Grollman,‘A, Am j physiol215 (1968) 15. Varotr, S, Nomura, J & Shooter, E M, Proc natl acad sci 57 (1967) 1782. 16. - Biochemistry 6 (1967) 2202. Received March 17, 1969

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