David A. Stanley, Dept. of Medicine, Stanford University School of Medicine, Palo Alto, CA.
John H. Frenster, Dept. of Medicine, Stanford University School of Medicine, Palo Alto, CA.
Demetrios A. Rigas, Div. of Experimental Medicine, University of Oregon Medical School, Portland, OR. 

INTRODUCTION

Human lymphocytes are capable of drastic metabolic (1) and morphologic (2) changes when exposed to certain glycoproteins such as phytohemagglutinin (PHA) in vitro. These changes can begin as early as 15 minutes after the addition of PHA (3, 4), and evolve in a predictable sequence during the next 72 hours; they include increased RNA and protein synthesis (5), conversion of heterochromatin to euchromatin (2), hypertrophy of the nucleolus and cytoplasm (6), the onset of DNA synthesis (7), and finally cell division (8). The occurence of these changes has permitted the use of PHA-activated human lymphocytes for studies of the human karyotype (9), the cell-mediated basis of immunity (10), the kinetics of lymphocyte proliferation (7), and aspects of reversible cell differentiation (11). Most recently PHA-activated autologous lymphocytes have been utilized for human cancer immunotherapy (12).

The localization of PHA within responsive lymphocytes, and the mechanism of its activation of lymphocytes, is not established. Radioautography of ³H-PHA within lymphocytes by light microscopy has suggested a localization within the cell nucleus (13), but because smears rather than sections were studied, cytoplasm overlying the nucleus could not be excluded. Immunofluoroescent studies of PHA localization have been ambiguous for the same reason (14). Lysosomes within lymphocytes have been found to release their contained hydrolytic enzymes during PHA activation (15), but isolated lysosomes are resistant to PHA (15). Plasma membrane permeability is altered during PHA activation (15), and a great variety of materials can prevent PHA activation by competition with PHA on the plasma membrane (16, 17), but these may only be affecting the entrance of PHA into the cell.

Recent analysis and purification of active PHA has revealed it to be a glycoprotein consisting of eight different sub-units associated together in an octamer molecule of molecular weight 138,000 (18). The availability of this purified glycoprotein octamer has permitted high-resolution localization by electron microscopy of the tritiated form of this PHA within human lymphocytes and monocytes. A partial account of this study has been presented (19).

METHODS

Tritiation of Phytohemagglutinin:

Twenty-four mg. of highly purified PHA in the form of the glycoprotein octamer (18) was exposed for 2 weeks to 15.0 curies of tritium gas at a pressure of 660 mm. Hg and a temperature of 26^oC. The resultant ³H-PHA was lyopholized from 20 ml. of distilled water, and possessed a specific activity of 1.4 millicuries/milligrams. When assayed within 2 weeks following the completion of tritiation, the mitogenic activity was only slightly reduced compared to the pre-tritiation mitogenic activity.

Incubation Conditions:

A modification of the the method of Moorehead, et al (9) was employed. Normal human blood was aspirated during the fasted state under sterile conditions into heparinized syringes, was thoroughly mixed with heparin, 4 units/ml, was centrifuged at 10 g. for 10 minutes, and the resultant leukocyte-rich supernate was collected and diluted 1:5 with medium 199. Additional heparin was added to a concentration of 4 units/ml, penicillin and streptomycin were each added to 0.060 mg/ml, and ³H-PHA (described above) was to 0.010 mg/ml, 5 ml aliquots of the resultant cell suspension, containing approximately 10⁶ lymphocytes/aliquot, were transferred to 15 ml screw-cap plastic flasks. These were tightly sealed, placed flat on their sides, and incubated at 37^oC for periods of time ranging from 15 minutes to 24 hours.

Electron Microscopy:

At the conclusion of incubation, the contents of each flask were centrifuged at 200 g for 10 minutes, and 5 mg of 5 percent glutaraldehyde dissolved in medium 199 was added to the resulatant centrifuge pellet. These were allowed to stand at 4^oC for 24 hours, and were then post-fixed in 1 percent OsO₄, dehydrated in progressive ethanol concentrations, embedded in epon, uniformly sectioned at 1000 A^o thickness, stained with uranyl acetate, and prepared for high resolution radioautography by the method of Caro and Van Tubergen (20). After 2 months exposure to the Ilford 4 emulsion, the grids were examined without NaOH removal of the developed emulsion on a Siemens Elmiskope 1A at 80 KV. The resultant micrographs were printed on a low-contrast photographic paper to enhance radioactive grain contrast.

RESULTS

Radioautographic electron micrographs of human lymphocytes, monocytes, and granulocytes cultured for either 15 minutes or 24 hours with ³H-PHA were examined. Over 300 separate cells were analyzed, and only those cells which were free of obvious signs of necrosis, and had evident active surface villous projections were included in the quantitation. On these criteria, granulocytes at 24 hours invariably showed necrotic changes and could not be analyzed, while even granulocytes cultured for only 15 minutes frequently showed either early necrosis or reduced villous projections.

Representative cells included in the analysis are illustrated in Figures 1 and 2. Each has active surface villous projections, a lack of evident necrotic changes, and a morphology typical of transforming lymphocytes or of monocytes in culture (2).


Fig. 1a, b, c, and d. Electron microscopic radioautographs of human lymphocytes after 24-hour incubation with ³H-PHA. The cells show surface villous projectiona and are free of obvious necrotic changes. The grains are localized chiefly within the nucleus of the lymphocytes. (Magnification: 10,000 X).

Fig. 2a, and b. Electron microscopic radioautographs of human monocytes after 24-hour incubation with ³H-PHA. The cells show surface villous projections and are free of obvious necrotic changes. The grains are localized chiefly within the cytoplasm of the monocytes. (Magnification: 5,000 X).

A preliminary analysis was done to determine the intensity of cell localization of ³H-PHA. Grains localized to the cell surface and interior of the cell were contrasted to external grains found within one cell diameter distance from the cell, an area 8 times as great as the cell area visualized on the micrograph.
 

TABLE 1. Intensity of Localization of ³H-PHA in Radioautographs.

	15 minutes:		24 hours:
	Lymphocytes	Monocytes	Lymphocytes	Monocytes
No. of cells analyzed:	7	6	25	9
No. of grains within cells:	31	38	115	85
No. of grains in 8-fold External Area:	18	17	63	3
Localization factor: (Internal/External)	13.8	17.8	14.6	226.6

The analysis (Table 1) revealed a greater than 13-fold localization of ³H-PHA by incubated cells compared to the immediate external environment of the cell, as reflected in the final radioautograph.

A detailed analysis was then made of the sites within the cells where radioactive grains could be resolved to specific intracellular organelles and structures.
 

TABLE 2. Subcellular Localization of ³H-PHA.

	15 minutes:		24 hours:
	Lymphocytes	Monocytes	Lymphocytes	Monocytes
No. of cells analyzed:	15	20	48	32
No. of grains touching plasma membrane:	20	21	43	67
No. of grains within cytoplasm:	26	70	67	279
No. of grains touching nuclear membrane:	8	7	38	33
No. of grains within  nucleus:	17	23	100	68
Mean No. of grains/cell:	4.7	6.0	5.2	13.9

The analysis (Table 2) revealed that after 15 minutes of incubation with ³H-PHA both lymphocytes and monocytes displayed a significant uptake, chiefly localized to the plasma membrane and cytoplasm, but with a significant quantity also localized to the interior of the nucleus. After 24 hours of incubation with ³H-PHA, lymphocytes displayed little further increase in mean uptake/cell, but did display an an altered distribution of ³H-PHA, with a majority of grains now found within the interior of the nucleus. By contrast, at 24 hours monocytes had continued to increase mean uptake/cell, and the majority of grains continued to be localized within the cytoplasm (Table 2). 

This analysis revealed that both lymphocytes and monocytes displayed a majority of their nuclear grains within the condensed heterochromatin of the nucleus, both after 15 minutes and after 24 hours of incubation with ³H-PHA.

This localization of ³H-PHA within the condensed heterochromatin portion of the nucleus was then compared to the intranuclear localization of other exogenous ligands which are being studied in this laboratory.
 

TABLE 4. Nuclear Binding Sites for Exogenous Ligands.

Ligand	Effect on DNA Template	Nuclear Binding Site	Reference
PHA	De-Repression	Heterochromatin	(19)
HgCl2	De-Repression	Heterochromatin	(21)
Acridine Orange	Inhibition	Euchromatin	(22)
Actinomycin	Inhibition	Euchromatin	(23)

Each of these ligands has a profound stimulatory or inhibitory effect on DNA template function within intact cells, and such a comparative analysis (Table 4) revealed that those ligands which are known to increase the DNA template synthesis of RNA (PHA (5), HgCl₂ (24)) are found to localize largely within condensed heterochromatin, while those ligands which are known to decrease the DNA template synthesis of RNA (Acridine Orange (25), Actinomycin (26)) are found to localize largely within extended euchromatin of intact cells.

DISCUSSION

The present data indicate that when ³H-PHA is incubated with living human lymphocytes and monocytes, an extensive penetration of the radioactive label into the interior of the cells can be demonstrated by radioautography of ultrathin sections of these cells. It is not known if such penetrated label represents metabolized or intact ³H-PHA, but such label is found within condensed heterochromatin, a cellular organelle which does not demonstrate uptake of such metabolic products as amino acids or nucleosides within intact cells or isolated nuclei (27). Therefore, the localization of ³H-PHA label within condensed heterochromatin suggests that, at a minimum, the ³H-PHA has not been metabolized to constitiuent amino acids.

Although monocytes display a greater uptake of ³H-PHA than do lymphocytes, a majority of the label within monocytes is confined to the cytoplasm, while lymphocytes display a majority of the label in the nucleus after 24 hours of incubation. Monocytes have a more extensively developed cytoplasm than do lymphocytes, including a larger number of lysosomes (15), and such lysosomes could play a defensive role in sequestering exogenous ligands such as PHA rather than allowing such ligands to penetrate into the nucleus. In the present study it was not possible to determine what proportion of cytoplasmic grains was confined to lysosomes, but there is considerable evidence for lysosomal sequestration of PHA (15).

The localization of ³H-PHA within the condensed heterochromatin of transforming lymphocytes is interesting in relation to the early ultrastructural changes which are observed within heterochromatin during lymphocyte transformation (2). The earliest changes consist of tunneling and conversion of condensed heterochromatin into extended euchromatin (2), and it seems reasonable that ³H-PHA localized within heterochromatin may be playing a role in such a transformation.

In this regard, it is necessary to compare the intracellular binding sites of ³H-PHA with other exogenous ligands. Both Acridine Orange and Actinomycin are capable of binding to DNA only when that DNA is comparatively free of overlying histones (28, 29), and for this reason such ligands are localized within extended euchromatin (22, 23). By contrast, both ³H-PHA and HgCl₂ are localized within heterochromatin, where histones are more available for reactivity (30). This might suggest that both ³H-PHA and HgCl₂ bind to histones. This result of such binding could be a displacement of the bound histones off from the underlying DNA templates, and an activation of RNA synthesis on such freed DNA templates (30).

Studies on cell-free systems to test this possibility are currently in progress.

SUMMARY

The highly-purified glycoprotein octamer of phytohemagglutinin was tritiated and incubated with human lymphocytes and monocytes for periods ranging from 15 minutes to 24 hours. Electron microscopic radioautography revealed a predominately cytoplasmic localization in monocytes, but a localization within the condensed heterochromatin of lymphocytes.

ACKNOWLEDGMENTS

We wish to thank Marie A. Shatos and Albert A. Keshgegian for their technical assistance.

SUPPORT

Supported in part by grants CA-10174 and AM-01006 from the National Institutes of Health, and by a contract at (45-1) -581 from the Atomic Energy Commission. JHF is a Research Scholar of the Leukemia Society.

REFERENCES

1. Rubin AD, and Cooper HL, "Evolving Patterns of RNA Metabolism during Transition from Resting State to Active Growth in Lymphocytes Stimulated by Phytohemagglutinin", Proc. Natl. Acad. Sci. U.S. 54, 469 (1965).

2. Tokuyasu K, Madden SC, and Zeldis LJ, "Fine Structural Alterations of Interphase Nuclei of Lymphocytes Stimulated to Growth Activity In Vitro", J. Cell Biol. 39, 630 (1968).

3. Killander D, and Rigler R, "Activation of Deoxyribonucleoprotein in Human Lymphoctes Stimulated by PHA. I. Kinetics of Binding of Acridine Orange to Deoxyribonucleoprotein", Exp. Cell Res. 54, 163 (1969).

4. Pogo BGT, Allfrey VG, and Mirsky AE, "RNA Synthesis and Histone Acetylation during the Course of Gene Activation in Lymphocytes", Proc. Natl. Acad. Sci. U.S. 55, 805 (1966).

5. Cooper HL, "RNA Metabolism in Lymphocytes Stimulated by Phytohemagglutinin. II. Rapidly Synthesized RNA and the Production of Ribosomal RNA", J. Biol. Chem. 243, 34 (1968).

6. Inman DR, and Cooper EH, "Electron Microscopy of Human Lymphocytes Stimulated by Phytohemagglutinin", J. Cell Biol. 19, 441 (1963).

7. Sasaki MS, and Norman A, "Proliferation of Human Lymphocytes in Culture", Nature 210, 913 (1966).

8. Nowell PC, "Phytohemagglutinin: An Initiator of Mitosis in Cultures of Normal Human Leukocytes", Cancer Res. 20, 462 (1960).

9. Moorhead PS, Nowell PC, Mellman WJ, Battips DM, and Hungerford DA, "Chromosome Preparations of Leukocytes Cultured from Human Peripheral Blood", Exp. Cell Res. 20, 613 (1960).

10. Oppenheim JJ, "Relationship of In Vitro Lymphocyte Transformation to Delayed Hypersensitivity in Guinea Pigs and Man", Fed. Proc. 27, 21 (1968).

11. Polgar PR, Kibrick S, and Foster JM, "Reversal of PHA-Induced Blastogenesis in Human Lymphocyte Cultures", Nature 218, 596 (1968).

12. Frenster JH, and Rogoway, WM, "In-Vitro Activation and Re-Infusion of Autologous Human Lymphocytes", Lancet 2, 979 (1968).

13. Conrad RA, "Autoradiography of Leukocytes Cultured with Tritiated Bean Extract", Nature 214, 709 (1967).

14. Razavi L, "Cytoplasmic Localization of Phytohemagglutinin in Peripheral White Cells", Nature 210, 444 (1966).

15. Hirschorn R, Brittinger G, Hirschorn K, and Weissmann G, "Studies on Lysosomes. XII. Redistribution of Acid Hydrolases in Human Lymphocytes Stimulated by Phytohemagglutinin", J. Cell Biol. 37, 412 (1968).

16. Borberg H, Yesner I, Gesner B, and Silber R, "The Effect of N-acetyl D-galactosamine and Other Sugars on the Mitogenic Activity and Attachment of PHA to Tonsil Cells", Blood 31, 747 (1968).

17. Pogo BGT, "The Effect of N-acetyl D-galactosamine Stimulated with Phytohemagglutinin", J. Cell Biol. 40, 571 (1969).

18. Rigas DA, and Head C, "The Dissociation of Phytohemagglutinin of Phaseolus Vulgaris by 8.0 M Urea and the Separation of the Mitogenic from the Erythroaggluntinating Activity", Biochem. Biophys. Res. Comm. 34, 633 (1968).

19. Stanley DA, Frenster JH, and Rigas DA, "Subnuclear Localization of Tritiated Phytohemagglutinin during Gene De-repression within Human Lymphocytes", J. Cell Biol. 39, 129a (1968).

20. Caro LG, and Van Tubergen RP, "High-Resolution Autoradiography. I. Methods", J. Cell Biol. 15, 173 (1962).

21. Frenster JH, "Ultrastructural Effects of Mercuric Chloride on Nuclear Heterochromatin within Human Lymphocytes", J. Cell Biol. 43, 39a (1969).

22. Frenster JH, "Electron Microscopic Localization of Acridine Orange Binding to DNA within Human Leukemic Bone Marrow Cells", Cancer Res. 31, 1128 (1971).

23.Harbers E, and Vogt M, "Studies on the Properties of Nucleohistones", in: "The Cell Nucleus. Metabolism and Radiosensitivity", (Klouwen HM, ed.), London: Taylor & Francis, Ltd. 1966.

24. Pauly JL, Caron GA, and Suskind RR, "Blast Transformation of Lymphocytes from Guinea Pigs, Rats, and Rabbits Induced by Mercuric Chloride In Vitro", J. Cell Biol. 40, 847 (1969).

25. Hurwitz J, Furth JJ, Malamy M, and Alexander M, "The Role of DNA in RNA Synthesis. III. The Inhibition of the Enzymatic Synthesis of RNA and DNA by Actinomycin D and Proflavin", Proc. Natl. Acad. Sci. U.S. 48, 1222 (1962).

26. Reich E, Franklin RM, Shatkin AJ, and Tatum EL, "Action of Actinomycin D on Animal Cells and Viruses", Proc. Natl. Acad. Sci. U.S. 48, 1238 (1962).

27. Frenster JH, Allfrey VG, and Mirsky AE, "Repressed and Active Chromatin Isolated from Interphase Lymphocytes", Proc. Natl. Acad. Sci. U.S. 50, 1026 (1963).

28. Rigler R, and Killander D, "Activation of Deoxyribonucleoprotein in Human Leukocytes Stimulated by PHA. II. Structural Changes of Deoxyribonucleoprotein and Synthesis of RNA", Exp. Cell Res. 54, 171 (1969).

29. Rigler R, Killander D, Bolund L, and Ringertz NR, "Cytochemical Characterization of Deoxyribonucleoprotein in Individual Cell Nuclei", Exp. Cell Res. 55, 215, (1969).

30. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of RNA", Nature 206, 680 (1965).

CONFERENCE DISCUSSION

Peter R. Polger, Boston, Massachusetts: Have you actually followed the label through the cell into the nucleus?

John H. Frenster: If you remember the data contrasting the 15-minute incubation with the 24-hour incubation, the majority of the grains in the lymphocytes were in the cytoplasm and on the plasma membranes, that majority being about 70%, at least, of the lymphocyte grain. By 24 hours the majority of the grains were found within the nucleus, and that majority approaching 70% as well, so that on that time course basis one must consider the hypothesis that there is transport from the plasma membrane through the cytoplasm to the nucleus with nuclear accumulation. That is quite evident in the 24 hours.

K. Lindahl-Kiessling, Uppsala, Sweden: I would like to know if your label will follow the chromatin also during mitosis so you could localize it over the metaphase idiogram. The reason is that we have treated lymphocytes with high specific activity ³H-uridine pulses before and after PHA stimulation and then made an attempt to localize the resulting chromosome breaks on the different chromosome types and on the individual segments of each type. The results do not indicate that areas which are conventionally described as heterochromatic, e.g., the centromeric region, have an increased incidence of breaks which you would expect if they were the primary point of attack, as you suggest.

John H. Frenster: That is very interesting data. We have not followed the incubations longer than 24 hours, but I think that might be very worthwhile doing. In that case one might want to use light rather than electron microscopy to look at the chromosomes. Were you defining heterochromatin by the apparent absence of uridine labeling?

K. Lindahl-Kiessling, Uppsala, Sweden: No.

John H. Frenster: I mention this because of the recent work of Tokuyasu. Electronmicroscopic changes that accompany PHA transformation suggest that whereas the nonstimulated lymphocyte may have 70-80% of its DNA in the heterochromatic form, in the PHA stimulated cell by 48 hours not more than 10% remains heterochromatic. It is interesting whether or not it is always going to remain the same 10% in different populations. I think that one would have to ask with that marked degree of heterochromatic change what is the significance of any persisting heterochromatin that might be evident in the karyotype for the labeling studies you suggested.

Martin Janis, Madison, Wisconsin: What percentage of your cells behave like the cells you have shown us and do all labeled cells appear to be undergoing early blastic change?

John H. Frenster: It depends on whether we are speaking of lymphocytes, monocytes or granulocytes. Granulocytes are very avid for PHA especially at 15 minutes, but even at 15 minutes they begin to show early necrotic changes. We have adopted the following criteria for including cells in our quantitative analysis: (1) that they show no necrotic change and (2) that they show extensive surface villi on their cell membranes. Using these criteria few if any granulocytes could be included. Almost 100% of monocytes will show some uptake and about half the lymphocytes will show uptake even as late as 24 hours. Now that figure has to be qualified by the recognition that our sections are only 1000 A^o thick. Fifty sections would encompass one nucleus, so that many of the cells we are calling negative are probably in fact positive if we could study their serial sections. But it seems as though 100% of the monocytes and up to 50% of the lymphocytes show the label as early as 15 minutes.

Alan Rosenthal, Bethesda, Maryland: I have two questions - one is a technical question. In your determinations of grain counts, you express your data as grain counts per structure; however, structures occupy varying percentages of the cell profile and the specific activity in one area may vary greatly. Now if the activity at the proposed site of localization is heterochromatin, your grains for that area should be greater than grains per unit area of euchromatin. I was wondering if you had data on such grain count specific activity. Secondly, how much of your radioactivity at various times of incubation are still chemically phytohemagglutinin? Is there any metabolism; if so, your disintegrations may not represent the original compound you started with.

John H. Frenster: The second question first. We are very much interested in how PHA is metabolized and we are trying to recover from incubated cells radioactive PHA or its metabolic products, but that work hasn't reached any significant point yet. Your point is well taken regarding organelle areas, and for that reason we did not choose to make quantitative statements about the relative incorporation into hetero- vs. euchromatin. I would only wish to emphasize that with time there is this change in distribution and that it's true for the lymphocytes and not for the monocytes. If one actually does a quantitative analysis for hetero- vs. euchromatin, the preference of 10 to 1 which we see in the grain count cannot be explained by the area which is available. There is a 3 to 1 preference in favor of hetero- so that is disproportionate localization. I think that the significant thing is that the lymphocytes seem to change with time; the monocytes do not. One could speculate that monocytes could be protected against nuclear penetration by their larger number of cytoplasmic lysosomes. That would be an interesting point - why doesn't the label get to the monocyte nucleus at all?

ADDITIONAL REFERENCES

0. Electron Microscopy of Human Lymphocytes before and after Activation by PHA (Busch H, 1974).

1. Keshgegian AA, Meisner LF, and Frenster JH, "Thymidine Reversal of Ribothymidine Inhibition of Lymphocyte Mitosis", in "Proc. Fourth Annual Leukocyte Culture Conf. 1969", (McIntyre OR, ed.), (1971), pp. 361-366, Apppleton-Century-Crofts, New York.

2. Frenster JH, and Rogoway, WM, "Immunotherapy of Human Neoplasms with Autologous Lymphocytes Activated In-Vitro", in "Proc. Fifth Annual Leukocyte Culture Conf.", (Harris J, ed.), (1970), pp. 359-373,
Academic Press, New York.

3. Frenster JH, "Phytohemagglutinin-Activated Autochthonous Lymphocytes for Systemic Immunotherapy of Human Neoplasms", Ann. N.Y. Acad. Sci. 277: 45-51 (1976).

4. Stanley DA, Frenster JH, and Rigas DA, "Subnuclear Localization of Tritiated Phytohemagglutinin during Gene De-repression within Human Lymphocytes", J. Cell Biol. vol. 39, part 2, p. 129a (1968).