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The in vitro effect of neuraminidase on human lymphocytes
JOURNAL
OF SURGICAL
The
RESEARCH
In vitro M.
Effect WAYNE
15, 9699 (1973)
of Neuraminidase FLYE, AND
M.D., D.
EMILY
BERNARD
PH.D.,
M.D.
MATERIALS
AND
METHODS
Preparation of lymphocytes. Heparinized blood was drawn from normal donors and sedimented with Plasmagel (Laboratoire Roger Bellon, Neuilly, France). A 95-99s purified lymphocyte preparation was obtained after passing the plasma through a nylon column and lysing the red cells with Tris ammonium chloride [3]. Cells were then incubated with (a) Vibrio cholera neuraminidase at a concentration of 4 units/l x lo6 cells for 20 min at 37OC (An), (b) Mitomycin-C (Sigma, St. Louis, MO.) 25-50 pg/l X lo6 cells for 20 min at 37% (Am) [2] (c) neuraminidase followed by mitomycin (Anm) or (d) calcium saline (A) [12]. All cells were then washed twice with calcium saline. 96
Press, Inc. form reserved.
REISNER,
Lymphocytes
means by which enzyme treatment produces these effects is not known. We have used the MLC to test for stimulation between neuraminidase treated and untreated cells from the same donor. Several other groups have previously used the MLC to test for autologous stimulation. Leukemia associated antigens were shown to stimulate autologous lymphocytes [l, 91 and cells from an established lymphocytoblast line stimulated the peripheral lymphocytes of the original normal donor [ll]. Autologous stimulation of normal lymphocytes has also been demonstrated following neuraminidase treatment [7, 171. In addition we have investigated the possibility that neuraminidase itself may have a stimulatory effect.
From the Department of Surgery and the Division of Immunology, Duke University Medical Center, Durham, North Carolina. Supported by National Institutes of Health Grants 5-TOl-GM-1709 and 5-ROl-A108897. Submitted for publication October 25, 1972.
@ 1973 by Academic of reproduction in any
G. AMOS,
LYMPHOCYTES ISOLATED FROM THE PERIPHERAL BLOOD of human subjects can be stimulated in vitro to undergo blast transformation. These blast cells are characteristically large pyroninophilic cells that actively synthesize nucleic acids and undergo mitosis. Such stimulation can be induced by a variety of specific and nonspecific including tuberculin agents [20], staphlococci filtrate [ 151, tetanus toxoid, diphtheria toxoid, smallpox vaccine [6], phytohemagglutinin [ 191, pokeweed mitogen [8] and enzyme treatment [ 181. Mixtures of lymphocytes from two genetically different individuals also cause mutual stimulation [16]. Unidirectional stimulation can be measured by inactivation of one cell population by treatment with mitomycin C [2]. Treatment with neuraminidase, an enzyme derived from the bacterium Vibrio cholerae, is known to enhance the immunogenicity of normal tissues [22] and tumors [5, 21, 261 and to increase the reactivity of lymphocytes in the cytotoxicity test [12]. Such treatment also appears to increase the ability of lymphoid cells, and fibroblasts to stimulate allogenic lymphocytes in one-way mixed leucocyte cultures (MLC) [16]. Watkins [26] has also reported activation of host lymphocytes by neuraminidase treated cancer cells. The
Copyright All rights
on Human
FLYE,
REISNER
AND
AMOS:
EFFECT
OF
NEURAMINIDASE
Method of culture. Cells for culture were suspended at a concentration of 1 X lo6 cells/ml in RPM1 1640 with 0.02 mM/ml glutamine (Gibco, Grand Island, New York), ampicillin 0.1 mg/ml and supplemented with 10% heat inactivated AB serum. For one-way MLC, 0.5 ml of the stimulating cells [5 X lo5 cells (Anm)] were mixed with an equal volume and number of responding cells (A) in 12 X 75 mm disposable glass tissue culture tubes with loose-fitting metal caps and incubated at 37% in an atmosphere of 5%C02 in air. Harvest. Triplicate cell cultures were labeled on different days by the addition of 1 PC of tritiated thymidine (Specific activity: 3Ci/mm, Schwarz/Mann, Orangeburg, N.Y.). Twelve hours later each cell culture was washed 3 times with cold saline, dissolved in 0.5 ml NCS solubilizer and rinsed with 10 ml scintilation fluid (0.0379 g POPOP, 22.74 g PPO in 3.79 liters of toluene) into vials. The samples were counted in a Packard Tricarb Scintillation Counter (Packard). The incorporation of tritiated thymidine was expressed as counts per minute (cpm) and the means of triplicate cultures were calculated. Two-way stimulation also employed 1 X lo6 cells per culture. Neuraminidase. Vibrio cholerae neuraminidase (Behringswerke) was obtained in sterile l-ml ampules containing 500 units (1 unit releases 1 pg N-acetylneuraminic acid from an acid a-1-glycoprotein substrate at 37% in 15 min at pH 5.5). Neuraminidase was inactivated by heating for 10 min at 100°C. This has been shown to result in complete loss of activity when tested on N-acetyl neuraminosyllactose 1211. This preparation is reported to be free of proteinases, aldolose, and lecithinase C. To determine the stimulating capacity of the neuraminidase preparation, it was added to 1 X lo6 neuraminidase treated or untreated cells per culture in serial two fold dilution from 0.5 to 64 units. These cultures were labeled, harvested and counted just as for the MLC. Smears were also prepared from the cell cultures for autoradiography at the
ON
HUMAN
LYMPHOCYTES
97
time of harvest by coating slides wit’h NBT-2 emulsion (Kodak), developing for two weeks and counterstaining with Giemsa. RESULTS Cells treated with neuraminidase alone or with both neuraminidase and (An) mitomycin-C (Anm) stimulated untreated autologous cells from 4 of 10 persons, studied (Fig. 1). In most cases the control values for the neuraminidase treated cells were equal to or slightly higher than untreated controls. When increasing quantities of neuraminidase (up to 64 units) were added to both control cell cultures and cells pretreated with neuraminidase, increasing CPM resulted (Fig. 2). Greater concentrations on Days 5 and 6 resulted in falling counts probably due to the changes occuring in the volume of the culture medium by the addition of increasing volumes of the neuraminidase and by exhaustion of the culture media nutrients. Although the pH of the enzyme preparation itself is 5.5 the media was buffered so that the pH of the culture did not change from an initial value of 7.8-7.9 with the increasing amounts of neuraminidase. Autoradiography demonstrated that 3-4s of neuraminidase stimulated cells were labelled while less than 1% of control cells were labelled. Since neu-
2500r ‘y zoooz .z : 15002 I: 8
IOOO-
A An ,,,....+b ,,..... ..‘,,‘na,, ‘p\ .v \ 7 ,...” /’ ‘.., ‘\ i..s’.’ ,d I’ * ..y.‘\ I. .,.,,,.. /c../ ,I .* 5’
‘\
........
7
-\.
500 -
DQys in Culture
Fig. 1. Tritiated thymidine uptake (cpm) in allogenic MLC in which A (control) An (Neuraminidase treated), and Anm (Neuraminidase + mitomycin treated) are mixed. (a-----0) (A---A.) AnAn, (V...V) AAn, AA, (W---m) AAnm.
98
JOURNAL
,_ -.-a-.0
0
-.-
OF
-.-.-&
05 I 2 Amount of Neurominidore
SURGICAL
Day 2 -.-. *-.-.--c
RESEARCH,
i -.---* . . .._._ c _ _ .
4 8 16 Used for Slimulalian
32 iUnits
64
8%~. 2. Response of human lymphocytes to increaskg amounts of neuraminidase. Day 2 (A----A), Day 3 (O-O), Day 5 (m -. * n ) and Day 6 (v---v).
raminidase is known to act on the glycoproteins of serum the full range of enzyme concentrations was preincubated with cells both before serum was added and with serum present. The difference in stimulation was insignificant. When heat inactivated enzyme was used no stimulation occurred. DISCUSSION There is definite MLC stimulation between neuraminidase treated and untreated autologous lymphocytes from some donors. Lundgren and Simmons [17] have recently reported similar findings as have Etheredge et al. [8]. The latter authors were able to increase the proportion of positive results by using varying numbers of stimulating cells and tritiated thymidine of higher specific activity (18 Ci/mM vs 3 Ci/mM in the present study). Gottschalk [lo] concluded from his studies with the enzymes of influenza virus and Vibrio cholerae that neuraminidase selectively splits a terminal sialic acid unit (N-acetylneuraminic acid). This involves a hydrolytic cleavage of the glycosidic bond joining the keto oxygen group of neuraminic acid to n-galactose or n-galactosamine, and possibly other sugars. We have found that neuraminidase itself can induce transformation as has been re-
VOL.
15,
NO.
2,
AUGUST
1973
ported for lymphocytes treated with agents such as chymotypsin, trypsin, papain [18], microwave irradiation [24], sonication [26] and antigen-antibody complexes in the presence of complement [13]. While there is no evidence to suggest a similar mechanism of activation in all these systems, Hirschhorn et al. [ 131 have suggested that membrane damage may be implicated as a common means of transformation. In the case of neuraminidase this appears to result from a specific enzymatic reaction. Turk et al. [25] observed blastoid alteration in lymphocytes subjected to sublethal sonication, but no increase in DNA or RNA synthesis rates. When Caso [4] added varying concentrations of neuraminidase to cultures of L929 fibroblasts, Hela cells and FL amnion cells an increase in both total DNA synthesis and the mitotic indices resulted. In this study neuraminidase also stimulated DNA synthesis in cultured lymphocytes as measured by scintillation counting and autoradiography as well as producing morphological changes. Kirchner [ 141 also demonstrated increased morphological blast transformation of the lymphocytes of some normal subjects when cultured with Vibrio cholera neuraminidase. The effects of neuraminidase on human lymphocytes appear to represent two different phenomenon. First, mixtures of normal lymphocytes and autologous neuraminidase treated lymphocytes are stimulated in MLC when low concentrations of enzyme are used. The MLC response is probably due to stimulation of the untreated cells by treated cells. This new antigenicity of treated lymphocytes may be explained by the unmasking of “new” antigens by simple removal of a carbohydrate coat or by surface conformational changes that allow previously “hidden” antigens to be recognized as foreign. Secondly when 15-20 times the amount of enzyme used to produce MLC stimulation is added to either enzyme treated or untreated lymphocytes another peak of stimulation results. This latter effect may represent nonspecific injury activation or may also result from uncovering
FLYE,
REISNER
AND
AMOS:
EFFECT
OF
NEURAMINIDASE
antigens on different populations of ceils and thereby setting up an internal MLC. Studies using neuraminidase and other enzymes show promise as a means of investigating the antigenic structure of cell surfaces that now is known to play such an important role in self recognition. This mechanism is a fundamental feature of t’he immune surveillance system for regulating tumor growth, viral replication and organ transplantation survival, REFERENCES Bach, M. L., Bach, F. H., and Joo, P. Leukemia-associated antigens in the mixed leultocyte culture test. Science 166:1520, 1969. 2. Bach, F., and Voynow, H. One-way stimulation in mixed leukocyte cultures. Science 1.
153:545,
1966.
Boyle, W. An extension of the ‘*Cr-release assay for the estimation of mouse cytotoxins. Transplantation 5:761, 1968. 4. Caso, L. V. Stimulation of DNA synthesis in mammalian cell cultures by receptor-destroying enzyme and neuraminidase. A,jznt. Rec. 172:551, 1972. 5. Currie, G. A., and Bagshawe, K. D. Tumour specific immunogenicity of methylcholanthrene-induced sarcoma celts after incubation in neuraminidase. &it. J. Cancer 23:141, 1969. 6. Elves, M. W., Roath, J., and Israels, M. C. G. The response of lymphocytes to antigen challenge in vitro. Lancet 1:806, 1963. 7. Etheredge, E. E., Shons, A. F., and Najarian, J. S. Neuraminidase-induced autologous stimulation of human leucocyte cultures. In M. R. Schwarz (ed.), Proceedings of the Sixth Leucocyte Culture Conference, p. 121. Academic Press, New York, 1972. 8. Fames, P., Barker, B. E., Brownhill, L. E., and Fanger, H. Mitogenic activity in phytolacca amcricana (pokeweed) Lancet 2:1100, 3.
1964. 9.
Fridman, W. H., and Kourilsky, F. M. Stimulation of lymphocytes by autologous leukaemic cells in acute leukaemia. Natrl)e 224~277,
10.
Il.
1969.
Gottschalk, A. Neuraminidaee : the specific enzyme of influenza virus and Vibrio cholerne. Biochem. Biophvs. Acta 23:645. 1957. Green, S. S., and Sell, K. W. Mixed leukocyte stimulation of normal peripheral leukocytes
ON
HUMAN
LYMPHOCYTES
99
by autologous Iymphoblastoid cells. Science 170:989, 1970. 12. Grothaus, E. A., Fly-e, M. W., Yunis, E., and Amos, D. B. Human lymphocyte antigenic reactivity modified by neuraminidase. Science 173:542, 1971. 13. Hirschhorn, K., Block-Shtacher, N., and Uhr, J. W. Stimulation of non-immunized human lymphocytes by antigen-antibody complexes. J. Clin. Invest. 46:1070, 1967. 14. Kirchner, H. The effect of neuraminidase on lymphocyte cultures. Lancet II :747, 1969. 15. Ling, N. R., Spicer. E., James, K., and Williamson, N. The activation of human peripheral lymphocytes by products of staphylococci. Brit. J. Haematol. 11:423, 1965. 16. Lundgram, G., Jeitz, L., Lundin, L., and Simmons, R. L. Increased stimulation by neuraminidase treated cells in mixed lymphocyte cultures. Fed. Proc. 30:395, 1971. G., and Simmons, R. L. Effect of 17. Lundgram, neuraminidase on the stimulating capacity of cells in human mixed lymphocyte cultures. Clin. Exp. Immunol. 9:915, 1971. 18. Mazzei, D., Nave, C., and Bazzi, C. Mitogenic action of papain. Lnncet 11:802, 1967. 19. Nowell, P. C. Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20:462, 1960. 20. Pearmain, G., Lycette, R. R., and Fitzgerald, I’. H. Tuberculin-induced mitosis in peripheral blood leukocytes. Lancet 1:637, 1963. 21. Sanford, B. H. An alteration in tumor histocompatibility. Tmnsplantation 5:1273, 1967. 22. Simmons, R. L., Rios, A., and Ray, P. I. Immunogenicity and antigenicity of lymphoid cells treated with neuraminidase. Nature (New Biol.) 231:179, 1971. 23. Steel, C. M., and Hardy, D. A. Evidence of altered antigenicity in cultured lymphoid cells from patients with infectious mononucleosis. Lnncet i:1322, 1970. 24. Stodolnik-Baranska. W. Lymphoblastoid transformation of lymphocytes in vitro after microwave radiation Natzcre 214:102, 1967. 25. Turk, A., Glade, P. R., and Charsin, L. N. Blast-l&c transformation induced in peripheral blood 1yml)hocytes by cellular injury. A comparison of sonicat ion and phyto-hemagglutinin. Blood 33:329, 1969. 26. Watkins, E., Jr., Ogata, Y., Anderson, L. L., and Watkins, E., III, and Waters, M. F. Activation of host lymphocytes cultures with cancer cells treated with neuraminidasc. Naf7cl.e (New Bioh.) 231:83, 1971.
OF SURGICAL
The
RESEARCH
In vitro M.
Effect WAYNE
15, 9699 (1973)
of Neuraminidase FLYE, AND
M.D., D.
EMILY
BERNARD
PH.D.,
M.D.
MATERIALS
AND
METHODS
Preparation of lymphocytes. Heparinized blood was drawn from normal donors and sedimented with Plasmagel (Laboratoire Roger Bellon, Neuilly, France). A 95-99s purified lymphocyte preparation was obtained after passing the plasma through a nylon column and lysing the red cells with Tris ammonium chloride [3]. Cells were then incubated with (a) Vibrio cholera neuraminidase at a concentration of 4 units/l x lo6 cells for 20 min at 37OC (An), (b) Mitomycin-C (Sigma, St. Louis, MO.) 25-50 pg/l X lo6 cells for 20 min at 37% (Am) [2] (c) neuraminidase followed by mitomycin (Anm) or (d) calcium saline (A) [12]. All cells were then washed twice with calcium saline. 96
Press, Inc. form reserved.
REISNER,
Lymphocytes
means by which enzyme treatment produces these effects is not known. We have used the MLC to test for stimulation between neuraminidase treated and untreated cells from the same donor. Several other groups have previously used the MLC to test for autologous stimulation. Leukemia associated antigens were shown to stimulate autologous lymphocytes [l, 91 and cells from an established lymphocytoblast line stimulated the peripheral lymphocytes of the original normal donor [ll]. Autologous stimulation of normal lymphocytes has also been demonstrated following neuraminidase treatment [7, 171. In addition we have investigated the possibility that neuraminidase itself may have a stimulatory effect.
From the Department of Surgery and the Division of Immunology, Duke University Medical Center, Durham, North Carolina. Supported by National Institutes of Health Grants 5-TOl-GM-1709 and 5-ROl-A108897. Submitted for publication October 25, 1972.
@ 1973 by Academic of reproduction in any
G. AMOS,
LYMPHOCYTES ISOLATED FROM THE PERIPHERAL BLOOD of human subjects can be stimulated in vitro to undergo blast transformation. These blast cells are characteristically large pyroninophilic cells that actively synthesize nucleic acids and undergo mitosis. Such stimulation can be induced by a variety of specific and nonspecific including tuberculin agents [20], staphlococci filtrate [ 151, tetanus toxoid, diphtheria toxoid, smallpox vaccine [6], phytohemagglutinin [ 191, pokeweed mitogen [8] and enzyme treatment [ 181. Mixtures of lymphocytes from two genetically different individuals also cause mutual stimulation [16]. Unidirectional stimulation can be measured by inactivation of one cell population by treatment with mitomycin C [2]. Treatment with neuraminidase, an enzyme derived from the bacterium Vibrio cholerae, is known to enhance the immunogenicity of normal tissues [22] and tumors [5, 21, 261 and to increase the reactivity of lymphocytes in the cytotoxicity test [12]. Such treatment also appears to increase the ability of lymphoid cells, and fibroblasts to stimulate allogenic lymphocytes in one-way mixed leucocyte cultures (MLC) [16]. Watkins [26] has also reported activation of host lymphocytes by neuraminidase treated cancer cells. The
Copyright All rights
on Human
FLYE,
REISNER
AND
AMOS:
EFFECT
OF
NEURAMINIDASE
Method of culture. Cells for culture were suspended at a concentration of 1 X lo6 cells/ml in RPM1 1640 with 0.02 mM/ml glutamine (Gibco, Grand Island, New York), ampicillin 0.1 mg/ml and supplemented with 10% heat inactivated AB serum. For one-way MLC, 0.5 ml of the stimulating cells [5 X lo5 cells (Anm)] were mixed with an equal volume and number of responding cells (A) in 12 X 75 mm disposable glass tissue culture tubes with loose-fitting metal caps and incubated at 37% in an atmosphere of 5%C02 in air. Harvest. Triplicate cell cultures were labeled on different days by the addition of 1 PC of tritiated thymidine (Specific activity: 3Ci/mm, Schwarz/Mann, Orangeburg, N.Y.). Twelve hours later each cell culture was washed 3 times with cold saline, dissolved in 0.5 ml NCS solubilizer and rinsed with 10 ml scintilation fluid (0.0379 g POPOP, 22.74 g PPO in 3.79 liters of toluene) into vials. The samples were counted in a Packard Tricarb Scintillation Counter (Packard). The incorporation of tritiated thymidine was expressed as counts per minute (cpm) and the means of triplicate cultures were calculated. Two-way stimulation also employed 1 X lo6 cells per culture. Neuraminidase. Vibrio cholerae neuraminidase (Behringswerke) was obtained in sterile l-ml ampules containing 500 units (1 unit releases 1 pg N-acetylneuraminic acid from an acid a-1-glycoprotein substrate at 37% in 15 min at pH 5.5). Neuraminidase was inactivated by heating for 10 min at 100°C. This has been shown to result in complete loss of activity when tested on N-acetyl neuraminosyllactose 1211. This preparation is reported to be free of proteinases, aldolose, and lecithinase C. To determine the stimulating capacity of the neuraminidase preparation, it was added to 1 X lo6 neuraminidase treated or untreated cells per culture in serial two fold dilution from 0.5 to 64 units. These cultures were labeled, harvested and counted just as for the MLC. Smears were also prepared from the cell cultures for autoradiography at the
ON
HUMAN
LYMPHOCYTES
97
time of harvest by coating slides wit’h NBT-2 emulsion (Kodak), developing for two weeks and counterstaining with Giemsa. RESULTS Cells treated with neuraminidase alone or with both neuraminidase and (An) mitomycin-C (Anm) stimulated untreated autologous cells from 4 of 10 persons, studied (Fig. 1). In most cases the control values for the neuraminidase treated cells were equal to or slightly higher than untreated controls. When increasing quantities of neuraminidase (up to 64 units) were added to both control cell cultures and cells pretreated with neuraminidase, increasing CPM resulted (Fig. 2). Greater concentrations on Days 5 and 6 resulted in falling counts probably due to the changes occuring in the volume of the culture medium by the addition of increasing volumes of the neuraminidase and by exhaustion of the culture media nutrients. Although the pH of the enzyme preparation itself is 5.5 the media was buffered so that the pH of the culture did not change from an initial value of 7.8-7.9 with the increasing amounts of neuraminidase. Autoradiography demonstrated that 3-4s of neuraminidase stimulated cells were labelled while less than 1% of control cells were labelled. Since neu-
2500r ‘y zoooz .z : 15002 I: 8
IOOO-
A An ,,,....+b ,,..... ..‘,,‘na,, ‘p\ .v \ 7 ,...” /’ ‘.., ‘\ i..s’.’ ,d I’ * ..y.‘\ I. .,.,,,.. /c../ ,I .* 5’
‘\
........
7
-\.
500 -
DQys in Culture
Fig. 1. Tritiated thymidine uptake (cpm) in allogenic MLC in which A (control) An (Neuraminidase treated), and Anm (Neuraminidase + mitomycin treated) are mixed. (a-----0) (A---A.) AnAn, (V...V) AAn, AA, (W---m) AAnm.
98
JOURNAL
,_ -.-a-.0
0
-.-
OF
-.-.-&
05 I 2 Amount of Neurominidore
SURGICAL
Day 2 -.-. *-.-.--c
RESEARCH,
i -.---* . . .._._ c _ _ .
4 8 16 Used for Slimulalian
32 iUnits
64
8%~. 2. Response of human lymphocytes to increaskg amounts of neuraminidase. Day 2 (A----A), Day 3 (O-O), Day 5 (m -. * n ) and Day 6 (v---v).
raminidase is known to act on the glycoproteins of serum the full range of enzyme concentrations was preincubated with cells both before serum was added and with serum present. The difference in stimulation was insignificant. When heat inactivated enzyme was used no stimulation occurred. DISCUSSION There is definite MLC stimulation between neuraminidase treated and untreated autologous lymphocytes from some donors. Lundgren and Simmons [17] have recently reported similar findings as have Etheredge et al. [8]. The latter authors were able to increase the proportion of positive results by using varying numbers of stimulating cells and tritiated thymidine of higher specific activity (18 Ci/mM vs 3 Ci/mM in the present study). Gottschalk [lo] concluded from his studies with the enzymes of influenza virus and Vibrio cholerae that neuraminidase selectively splits a terminal sialic acid unit (N-acetylneuraminic acid). This involves a hydrolytic cleavage of the glycosidic bond joining the keto oxygen group of neuraminic acid to n-galactose or n-galactosamine, and possibly other sugars. We have found that neuraminidase itself can induce transformation as has been re-
VOL.
15,
NO.
2,
AUGUST
1973
ported for lymphocytes treated with agents such as chymotypsin, trypsin, papain [18], microwave irradiation [24], sonication [26] and antigen-antibody complexes in the presence of complement [13]. While there is no evidence to suggest a similar mechanism of activation in all these systems, Hirschhorn et al. [ 131 have suggested that membrane damage may be implicated as a common means of transformation. In the case of neuraminidase this appears to result from a specific enzymatic reaction. Turk et al. [25] observed blastoid alteration in lymphocytes subjected to sublethal sonication, but no increase in DNA or RNA synthesis rates. When Caso [4] added varying concentrations of neuraminidase to cultures of L929 fibroblasts, Hela cells and FL amnion cells an increase in both total DNA synthesis and the mitotic indices resulted. In this study neuraminidase also stimulated DNA synthesis in cultured lymphocytes as measured by scintillation counting and autoradiography as well as producing morphological changes. Kirchner [ 141 also demonstrated increased morphological blast transformation of the lymphocytes of some normal subjects when cultured with Vibrio cholera neuraminidase. The effects of neuraminidase on human lymphocytes appear to represent two different phenomenon. First, mixtures of normal lymphocytes and autologous neuraminidase treated lymphocytes are stimulated in MLC when low concentrations of enzyme are used. The MLC response is probably due to stimulation of the untreated cells by treated cells. This new antigenicity of treated lymphocytes may be explained by the unmasking of “new” antigens by simple removal of a carbohydrate coat or by surface conformational changes that allow previously “hidden” antigens to be recognized as foreign. Secondly when 15-20 times the amount of enzyme used to produce MLC stimulation is added to either enzyme treated or untreated lymphocytes another peak of stimulation results. This latter effect may represent nonspecific injury activation or may also result from uncovering
FLYE,
REISNER
AND
AMOS:
EFFECT
OF
NEURAMINIDASE
antigens on different populations of ceils and thereby setting up an internal MLC. Studies using neuraminidase and other enzymes show promise as a means of investigating the antigenic structure of cell surfaces that now is known to play such an important role in self recognition. This mechanism is a fundamental feature of t’he immune surveillance system for regulating tumor growth, viral replication and organ transplantation survival, REFERENCES Bach, M. L., Bach, F. H., and Joo, P. Leukemia-associated antigens in the mixed leultocyte culture test. Science 166:1520, 1969. 2. Bach, F., and Voynow, H. One-way stimulation in mixed leukocyte cultures. Science 1.
153:545,
1966.
Boyle, W. An extension of the ‘*Cr-release assay for the estimation of mouse cytotoxins. Transplantation 5:761, 1968. 4. Caso, L. V. Stimulation of DNA synthesis in mammalian cell cultures by receptor-destroying enzyme and neuraminidase. A,jznt. Rec. 172:551, 1972. 5. Currie, G. A., and Bagshawe, K. D. Tumour specific immunogenicity of methylcholanthrene-induced sarcoma celts after incubation in neuraminidase. &it. J. Cancer 23:141, 1969. 6. Elves, M. W., Roath, J., and Israels, M. C. G. The response of lymphocytes to antigen challenge in vitro. Lancet 1:806, 1963. 7. Etheredge, E. E., Shons, A. F., and Najarian, J. S. Neuraminidase-induced autologous stimulation of human leucocyte cultures. In M. R. Schwarz (ed.), Proceedings of the Sixth Leucocyte Culture Conference, p. 121. Academic Press, New York, 1972. 8. Fames, P., Barker, B. E., Brownhill, L. E., and Fanger, H. Mitogenic activity in phytolacca amcricana (pokeweed) Lancet 2:1100, 3.
1964. 9.
Fridman, W. H., and Kourilsky, F. M. Stimulation of lymphocytes by autologous leukaemic cells in acute leukaemia. Natrl)e 224~277,
10.
Il.
1969.
Gottschalk, A. Neuraminidaee : the specific enzyme of influenza virus and Vibrio cholerne. Biochem. Biophvs. Acta 23:645. 1957. Green, S. S., and Sell, K. W. Mixed leukocyte stimulation of normal peripheral leukocytes
ON
HUMAN
LYMPHOCYTES
99
by autologous Iymphoblastoid cells. Science 170:989, 1970. 12. Grothaus, E. A., Fly-e, M. W., Yunis, E., and Amos, D. B. Human lymphocyte antigenic reactivity modified by neuraminidase. Science 173:542, 1971. 13. Hirschhorn, K., Block-Shtacher, N., and Uhr, J. W. Stimulation of non-immunized human lymphocytes by antigen-antibody complexes. J. Clin. Invest. 46:1070, 1967. 14. Kirchner, H. The effect of neuraminidase on lymphocyte cultures. Lancet II :747, 1969. 15. Ling, N. R., Spicer. E., James, K., and Williamson, N. The activation of human peripheral lymphocytes by products of staphylococci. Brit. J. Haematol. 11:423, 1965. 16. Lundgram, G., Jeitz, L., Lundin, L., and Simmons, R. L. Increased stimulation by neuraminidase treated cells in mixed lymphocyte cultures. Fed. Proc. 30:395, 1971. G., and Simmons, R. L. Effect of 17. Lundgram, neuraminidase on the stimulating capacity of cells in human mixed lymphocyte cultures. Clin. Exp. Immunol. 9:915, 1971. 18. Mazzei, D., Nave, C., and Bazzi, C. Mitogenic action of papain. Lnncet 11:802, 1967. 19. Nowell, P. C. Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20:462, 1960. 20. Pearmain, G., Lycette, R. R., and Fitzgerald, I’. H. Tuberculin-induced mitosis in peripheral blood leukocytes. Lancet 1:637, 1963. 21. Sanford, B. H. An alteration in tumor histocompatibility. Tmnsplantation 5:1273, 1967. 22. Simmons, R. L., Rios, A., and Ray, P. I. Immunogenicity and antigenicity of lymphoid cells treated with neuraminidase. Nature (New Biol.) 231:179, 1971. 23. Steel, C. M., and Hardy, D. A. Evidence of altered antigenicity in cultured lymphoid cells from patients with infectious mononucleosis. Lnncet i:1322, 1970. 24. Stodolnik-Baranska. W. Lymphoblastoid transformation of lymphocytes in vitro after microwave radiation Natzcre 214:102, 1967. 25. Turk, A., Glade, P. R., and Charsin, L. N. Blast-l&c transformation induced in peripheral blood 1yml)hocytes by cellular injury. A comparison of sonicat ion and phyto-hemagglutinin. Blood 33:329, 1969. 26. Watkins, E., Jr., Ogata, Y., Anderson, L. L., and Watkins, E., III, and Waters, M. F. Activation of host lymphocytes cultures with cancer cells treated with neuraminidasc. Naf7cl.e (New Bioh.) 231:83, 1971.