Experimental
CELL-CELL OF RAT
Cell Research
109 (1977) 211-221
INTERACTIONS
IN BLAST
LYMPHOCYTES
BY NEURAMINIDASE
GALACTOSE MARGARET Department
C. KIELIAN,
TRANSFORMATION AND
OXIDASE
CARL F. BEYER and WILLIAM
of Biochemical Cytology, The Rockefeller New York, NY 10021, USA
E. BOWERS
University,
SUMMARY Optimal reaction conditions for the in vitro blast transformation of rat lymph node cells (LNC) by neuraminidase (N) and galactose oxidase (GO) were determined. Treatment with either enzyme alone, or with GO before N, did not produce significant stimulation. The kinetics and magnitude of the response to NGO, as measured by [3H]TdR incorporation, were compared with those induced by periodate treatment and by concanavalin A (ConA). Rat LNC treated with NGO (but blocked by mitomycin C) caused blast transformation of untreated syngeneic LNC. The magnitude of this indirect response was about one-quarter of that attained for NGO-treated LNC, and the kinetics were somewhat slower. Supernatants from cultures of NGO-stimulated cells were not able to activate untreated cells, and, using Marbrook culture vessels, no evidence was found for the release of soluble factors that could either activate untreated cells or enhance the magnitude of indirect stimulation. Indirect stimulation, therefore, appears to require cell-cell contact. A second kind of cellular interaction was observed in exueriments in which NGO-treated LNC were cultured at different cell densities in the presence or absence of mitomycin C-blocked filler LNC. In the absence of tiller cells. the incornoration of PHlTdR was a linear function of the number of NGO-treated cells in each culture-over a wide range of cell densities. However, if tiller cells were present, [3H]TdR-incorporation was substantially enhanced. Thus the filler cell population contains cells that assist or potentiate the blast response to NGO, probably by increasing the number of lymphocytes that are stimulated
Novogrodsky & Katchalski first reported that sequential treatment of mouse spleen cells with neuraminidase (N) and galactose oxidase (GO) led to extensive blast transformation [l]. They suggested that removal of the terminal sialic acid residues from cell surface glycoproteins by neuraminidase revealed galactose and N-acetylgalactosamine residues [2], which were oxidized to aldehydes at the C6 positions by galactose oxidase. The function of the aldehyde groups in initiating blast transformation is unknown, but their importance is indicated by the fact that reagents that react with al-
dehydes, such as sodium borohydride or hydroxylamine, can inhibit transformation. An intriguing hypothesis is that the aldehydes form Schiff bases that cross-link molecules on the surface of the same or different cells [ 1, 31. One of the advantages of this mitogenic system is that it allows an evaluation of the role of cellular interactions in the blastogenie response. Since NGO treatment produces a chemical modification of the surface of the treated cells, mixtures of treated and untreated cells can be used to study the importance of cell interactions on the final &I,
Cdl
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109 (1977)
212
Kielian,
Beyer and Bowers
response. Two general classes of interacactivity was measured routinely [7] and was found to be consistently lower by -35% the value spetions between treated and untreated cells cified by the supplier; units in this paper refer to our can be considered: (1) the treated cells in- measured activity. After incubation, LNC were centriand washed once in PBS containing CaCI,. duce transformation of untreated cells, a fuged Periodate. LNC at a concentration of 2X 10’1 ml in process we have termed indirect stimulaice-cold PBS were mixed with an equal volume of 2.4 mM sodium metaperiodate in ice-cold PBS pretion [4]; and (2) the untreated cells influence pared sterilely immediately before use. The suspenthe transformation of the treated cells. sion was kept on ice with occasional gentle agitation for 15 min and then centrifuged at 535 g for 5 min at These interactions could involve cell-cell 4°C. The treated LNC were then washed once in contacts and/or the release of soluble me- HBSS at room temperature. Mitomycin C. LNC were prevented from subdiators. Both kinds of interactions have sequent division by incubating in the dark at 37°C for been observed previously in a mitogenic 30 min IO’ cells/ml in the presence of mitomycin C pg/ml, Sigma, St Louis, MO), in complete mesystem related to NGO, namely the genera- (25 dium consisting of RPM1 1640 (Associated Biomedic tion of cell surface aldehydes by periodate Svstems. Inc.. Buffalo. N.Y.) supplemented with 20% (56°C for 30 minjhorse serum (Grand oxidation of sialic acid residues [4, 5, 61. In heat-inactivated Island Biological Company, Grand Island, N.Y.). The the present study, we have determined the cells were centrifuged and washed once in HBSS. treated with both mitomvcin C and NGO were inreaction conditions for neuraminidase and LNC cubated first with mitomycin C, washed, and then galactose oxidase treatment which lead to treated with NGO. optimal stimulation of rat lymph node cells Cell culture conditions (LNC), and we have compared the kinetics With the exception of the cell culture conditions used and magnitude of the response to NGO for the experiment presented in table I, LNC were with those induced by periodate and by the suspended at a concentration of 3X 106/ml in complete medium. 1.0 ml of a cell suspension was cultured soluble lectin, ConA. We then determined in each well of a Multiwell tissue culture plate (no. the magnitude of indirect stimulation in this 3008, Falcon Plastics, Oxnard, Calif.) incubated at system and investigated whether the cel- 37°C and continuously gassed with a humidified atmosphere of 7 % CO, in air. For each point, triplicate lular interactions involved in this process or quadruplicate cultures were established. Cell viabilities, assessed by trypan blue exclusion, were were mediated by soluble factors. MATERIALS Preparation
AND METHODS
of lymph node cells
Cervical and mesenteric lymph nodes were removed from 150 to 250 g Lewis (LEW) rats (Microbiological Associates, Inc., Bethesda, Md), trimmed free of fat, rinsed three times with Hanks’ balanced salt solution (HBSS), and teased with dissecting needles in sterile HBSS. After rapid filtration through sterile cotton, the lymph node cells (LNC) were centrifuged at 375 g for 10 min and then washed twice in HBSS. Cell counts were made with a Coulter counter, Model B (Coulter Electronics, Inc., Hialeah, Fla).
Treatments and galactose oxidase (NGO). LNC at a concentration of 2xlO’/ml were incubated at 37°C for 30 min in Dulbecco’s phosphate-buffered saline (PBS) containing CaCl, (100 mg/l), 15 U/ml of Vibrio cholera neuraminidase (E.C. 3.2.1.18) (Calbiochem. La Jolla, Calif.) and 2.5 U/ml of Dactyl& dendroides galactose oxidase (E.C. 1.1.3.9) (Worthington Biochemical Corp., Freehold, N.J.). Galactose oxidase Neuraminidase
E.rp Cd
Rrs
determined at the start of the culture period and always exceeded 90 %. In one experiment LNC were incubated in the presence of 25 pg/ml of ConA (MilesYeda, Kankakee, Ill.).
109 (1977)
Labeling of cells with [3H]thymidine, cell harvesting, and preparation for scintillation counting With the exception of the experiments presented in figs 4 and 5, cells were labeled during the terminal 3 h of culture by adding to each well 25 ~1 of a concentrated labeline solution that uroduced a final concentration of 1 X 10mr M thymidine, 300 mCi/mmole of [6-3H]thymidine (SchwarzlMann, Orangeburg, N.Y.: 9 Ci/ mmole). At the end of the ct&ure period, the cells were collected on glass fiber filters (934AH, Reeve Anael. Clifton. N.J.) with the aid of a semi-automatic cell harvester ‘(M12k, Biomedical Research and Development Lab, Rockville, Md). When dry, the filters were placed in scintillation vials, 0.5 ml of Soluene (Packard, Downers Grove, Ill.) was added, and after a minimum of 2 h, 10 ml of a toluene based scintillation fluid containing 22.5 % Triton X-100 and 0.1% acetic acid was added. Radioactivity was measured in a Packard mode1 2002 liquid scintillation spectrometer.
Cell-cell
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in blast transformation
213
Table 1. [3H]Thymidine incorporation by rat lymph node cells treated with neuraminidase and galactose oxidase singly or sequentially [3H]Thymidine
incorporated”
at
1st treatment
2nd treatment
N”
GO’
90 465+2 (100.0)’
GO
N
3 727+74 (3.4)
2 809?63 (2.0)
2 607k581 (2.6)
N
-
983&43 (0.3)
936+336 (0.3)
659294
GO
-
I 355?348 (0.7)
925+59 (0.3)
832YclS3 (0.4)
-
705&239
585+94
4541112
60 h
48 h 757”
72 h
III 901*7 (100.0)
845
84 676&7 (100.0)
205
(0.2)
” During the last 2 h of culture 2 &i of [6iYH]thymidine (9 Ci/mmol) was added to each well of a Multiwell plate initially containing 0.6 ml of LNC (5x lO”/ml) in Dulbecco’s Modified Eagle’s medium supplemented with 20% heat-inactivated horse serum. ’ Fifty units of neuraminidase (N) incubated at 37°C for 30 min with 80x 10” LNC/ml of phosphate-buffered saline (lacking calcium). ’ 0.5 units of galactose oxidase (GO) incubated at room temperature for 30 min with 20x lo6 LNC/ml of phosphate-buffered saline (lacking calcium). ‘r Values given are means f S.D. for triplicate cultures. “ In parenthesis, the percent of [3H]thymidine incorporation relative to that of NGO treatment (100%); background (no treatment) subtracted from each value.
Culture and labeling Marbrook vessels
of cells in
Threaded, periscopic Marbrook vessels (Model B-9008) consisting of an inner, middle, and outer chamber nesting inside of each other were obtained from Bio-Research Glass, Inc. (Vineland, N.J.). Cells were cultured in the inner and middle chambers on membrane filters (Metricil TCM-450, pore size, 0.45 Frn; Gelman Instrument Company, Ann Arbor, Mich.) attached to the chambers by a Teflon O-ring and an annular plastic screw cap. The membrane filter for the inner chamber was 10.6 mm in diameter and for the middle chamber, 22 mm. The Marbrook vessels were prepared for cell culture by a modification of the procedure of Farrar [8]. After a thorough rinse of the chambers with deionized distilled water, chambers were assembled, filled with water, and autoclaved. On the day before use, water was replaced by RPM1 1640 containing 20% heatinactivated horse serum and the vessels were then incubated overnight at 37°C. After removal of the medium, LNC at a concentration of 5~ 106/ml in complete medium were added in a volume of 0.6 ml to the inner chamber and of 2.0 ml to the middle chamber. The outer chamber was filled with 20 ml of complete medium. Marbrook vessels were then incubated at 3PC in a humidified atmosphere of 7% CO, in air. After 44 h, [3H]thymidine was added to all three chambers to bring the radioactivity in each to 0.5 &i/ml, and the vessels were incubated an additional 8 h. At the end of the labeling period, the cells from each chamber were harvested separately onto glass fiber filters (Whatman
GF/C) in a multiple well filtering apparatus (Sampling Manifold no. 1225, Millipore Corporation, Bedford, Mass.). The filters were washed once with ice-cold HBSS, three times with 5 ml of ice-cold 5% trichloroacetic acid, and once with methanol. 0.5 ml of Soluene was added to each filter and radioactivity determined as above.
RESULTS Sequential enzyme treatment is required for LNC stimulation The results presented in table 1 show that extensive stimulation occurred only when rat LNC were treated first with neuraminidase and then with galactose oxidase. Reversal of this sequence or incubation with either enzyme alone was ineffective. Similar results were obtained at all three time points. Determination of optimal enzyme concentrations for LNC stimulation As shown in table 2, treatment of LNC simultaneously with neuraminidase and E.V/> cc// HcsIOY(1977)
2 14
Kielian,
Beyer and Bowers
Table 2. Determination of optimal conditions for simultaneous neuraminidase and galactose oxidase treatment of rat lymph node cells
creasing the galactose oxidase concentration while keeping neuraminidase at a fixed concentration led to a considerably higher degree of stimulation. Less stimulation was noted at concentrations of neuraminidase Neuraminidase Galactose oxidase cont. (U/ml) cont. exceeding 62.5 U/ml and of galactose ox0.1 0.5 2.5 5.0 10.0 idase above 2.5 U/ml, perhaps due to con(U/ml) taminating substances in the enzyme pre2.5 26.1” 52.3 70.1 parations. Commercial galactose oxidase 33.8 56.8 79.0 12.5 and neuraminidase may contain proteases 67.3” 43.4’ [9, lo], and we have observed that Cfo39.5d stridium perfringens neuraminidase (Wor15 91.0 thington) was toxic to LNC at low con62.5 34.9 90.3” 100.0’ 96.2 82.6 centrations. On the basis of these experi97.3’ 100.0” 93.8 72.1 80.9 80.4 ments, standard conditions of 15 U/ml of 82.5 86.1 70.1 Vibrio cholera neuraminidase 125 and 2.5 U/ml of galactose oxidase were chosen for subLNC were treated at 37°C for 30 min simultaneously with neuraminidase and galactose oxidase at the given sequent NGO treatment of LNC.
concentrations. Triplicate or quadruplicate cultures were established for each set of conditions. LNC were incubated for 53 h, labeled with [3H]thymidine, harvested, and prepared for scintillation counting as described in Materials and Methods. a Percent of maximum incorporation of [3H]thymidine. b Simultaneous enzyme treatment for 60 min at 37°C. c Separate enzyme treatment of 30 min each. ’ Lymph node cells treated simultaneously with neuraminidase and galactose oxidase in phosphate-buffered saline lacking calcium. e Results of two experiments.
galactose oxidase was as effective as sequential treatment with the two enzymes. Optimal conditions in this system were established by varying the concentration of enzymes incubated together with LNC at 37°C for 30 min, and the results of two experiments are given in table 2. Since maximal stimulation in both experiments occurred after treatment with 62.5 U/ml of neuraminidase and 2.5 U/ml of galactose oxidase, the other results have been normalized to these conditions. Only a modest increase in stimulation resulted when the galactose oxidase concentration was kept low (0.1 U/ml) and the concentration of neuraminidase was increased, whereas inExp Cdl
Res 109 (1977)
Kinetics of stimulation Aliquots of one preparation of rat LNC were treated with either NGO, periodate, or ConA, and the kinetics of the responses were compared (fig. 1). NGO- and periodate-treated cells showed similar kinetics
0
Fig. 1. Abscissa: time from start of culture (hours); ordinate: [3H]thymidine incorporation (cpmx 10m3). [3H]Thymidine incorporated at various times by LNC that were untreated (O-O) or treated with ConA (CD), NGO (A-A), or sodium periodate (0-O). Each point represents the average of quadruplicate cultures. Standard deviations were less than 13 % in all cases.
Cell-cell
interactions
215
in blast transformation
only half the cells in the mixture can spond, the [3H]thymidine incorporation the peak is about 25 % of that at the peak NGO-treated LNC. A value of 34% is tained by comparing the areas under [3H]thymidine incorporation curves.
reat for obthe
Stimulation at different ratios of NGO-treated to untreated LNC Owing to the important contribution made by indirect stimulation, the composition of 100 40 60 80 0 20 a cell mixture producing maximum [3H]thymidine incorporation was determined. Fig. 2. Abscissu: time from start of culture (hours): ordinafe: [3H]thymidine incorporation (cpmx 10e3). [3H]Thymidine incorporated at various times by un- At constant cell density, the ratio of NGO-, treated LNC (O-O), NGO-treated LNC (O-O), mitomycin C-treated LNC to untreated NGO-treated LNC blocked with mitomycin C (A-A), LNC was varied between the limits of the and a 1 : I mixture of untreated LNC and NGO-treated LNC blocked with mitomycin C (W-B). Each point pure component cell preparations. The represents the average of quadruplicate cultures. averaged results of two experiments, Standard deviations were less than 15% in all cases. presented in fig. 3, show that maximum incorporation of [3H]thymidine occurred in with peak incorporation occurring at 51 h, but the magnitude of the response induced by NGO was significantly greater. In contrast, cells stimulated with ConA showed peak incorporation of thymidine near 70 h, and the magnitude of the response was much greater than for either periodate or NGO. A more detailed kinetic curve for NGO stimulation of LNC is shown in fig. 2, which also includes the kinetics for a 1 : 1 mixture of untreated LNC and NGO-, mitomycin C-treated LNC. Kinetics similar to those already seen in fig. 1 were obtained for NGO-treated LNC, but different kinetics were found for the 1 : 1 mixture. Incorporation of [3H]thymidine peaked between 60 and 66 h and then decreased over the next 2 days. Separate cultures of untreated LNC and LNC doubly treated with NGO and mitomycin C showed at all times a significantly lower [3H]thymidine incorporation than their 1 : 1 mixture. Bearing in mind that
Fig. 3. Abscissa: fraction of untreated cells; ordinate: percent of maximal stimulation. Percent of maximum [3H]thymidine incorporated by mixtures of untreated LNC and NGO-, mitomycin Ctreated LNC which were co-cultured at different ratios but at a constant cell density. [3H]Thymidine was added 3 h before termination of the cultures at 66 h. The background contributed by the untreated LNC and doubly treated LNC to each mixture was subtracted, and the results of two experiments were normalized. Cpm at 100% for the two experiments were 35 000 and 40 000. The means and standard deviations are presented. Eup Cell
Re.7 109 (1977)
216
Kielian,
I@, 0
,
0.3
06
Beyer and Bowers
, 09
,
,
,
I.2
15
I8
, 21
, 24
, 27
, 30
Fig. 4. Abscissa:
responder cell density per culture incorporation (cpm x 10m3)per IO6 responder cells. r3H]‘I;hymidine -incorporated per IO6 NGO-treated LNC (responder cells) by cultures containing different numbers of NGO-treated LNC. Dilutions were made with medium (O-O), mitomycin C-treated LNC (O--O), or NGO-treated LNC blocked with mitomycin C (0-O). Each culture in which NGO-treated LNC were diluted with mitomycin C-treated or NGO-, mitomycin C-treated LNC had a constant cell density of 3 x lo6 cells. r3H]Thymidine was added 4 h before termination of the cultures at 64 h. Standard deviations are indicated in cases where they exceed the size of the symbol.
x 10m6; ordinate: [3H]thymidine
cultures comprised of 70-80% untreated LNC and 20-30 % LNC doubly treated with NGO and mitomycin C.
plete medium gave essentially the same results as found previously for periodate [4]; [3H]thymidine incorporation per lo6 cells was constant between 0.6~ IO” and 3X lo6 LNC/ml, but decreased at cell densities below 0.6~ IO6 LNC/ml. Interestingly, dilution of the same NGO-treated LNC with mitomycin C-treated LNC gave different results. As the proportion of mitomycin Ctreated LNC increased, the incorporation of [3H]thymidine per IO6 NGO-treated LNC also increased until a peak was reached which occurred in cultures containing 0.24~ lo6 NGO-treated LNC and 2.76~ lo6 mitomycin C-treated LNC. Further dilution with mitomycin C-treated LNC reduced the r3H]thymidine incorporation per lo6 NGOtreated LNC. When NGO-treated LNC were diluted with LNC that were treated with both NGO and mitomycin C, the shape of the curve was similar, but an even greater [3H]thymidine incorporation was found. Because the experiment presented in fig. 4 was performed at only one time point, the possibility existed that the differences in [3H]thymidine incorporation for NGOtreated LNC diluted with medium or with mitomycin C-treated LNC actually was due to kinetic differences in the two situations. As shown in fig. 5, the kinetics of proliferating cells at different densities were similar regardless of whether they were diluted with medium or with blocked syngeneic cells. At comparable densities of NGO-treated cells, the incorporation of r3H]thymidine was greater in cultures diluted with mitomycin C-treated cells than with medium.
Stimulation as a function of cell density The possibility that cell interactions may be involved in the response to NGO treatment was further explored by varying the density of NGO-treated LNC. Treated cells were diluted with complete medium or metabolically blocked syngeneic LNC. For the latter, a constant density of 3x IO6 cells per culture was maintained by diluting NGOtreated LNC with either mitomycin Ctreated LNC or LNC doubly treated with Attempt to demonstrate soluble factors in NGO stimulation NGO and mitomycin C. Three experiments yielded similar results; one is presented in The medium from cultures of NGO-treated fig. 4. LNC was tested for the presence of factors Dilution of NGO-treated cells with com- that produce or enhance blastogenesis. A E.rl,
Cdl
RP.\ 109 (1977)
Cell-cell
0
24
48
72
96
120
FI’g. 5. Abscissa: time from the start of culture (hours); ordinate: [3H]thymidine incorporation (cpmx lo-? per culture. Kinetics of [“Hlthymidine incorporation by cultures diluted with medium (O-O) or mitomycin C-treated LNC (O-Cl). Number of NGO-treated cells per culture was: (A) and (B) 1.8x106, (C) and (D) 0.6x10fi, and (E) 0.24~ IO”. Cultures containing NGO-treated LNC and mitomycin C-treated LNC had a constant cell density of 3 x IO@. [3H]Thymidine was added 4 h before termination of the cultures. Standard deviations are indicated in cases where they exceed the size of the symbol.
series of Petri dishes containing NGOtreated LNC or untreated LNC were cultured and at intervals thereafter, medium was taken from each of the two types of cul-
interactions
in blast transformation
217
ture, any contaminating cells removed by centrifugation, and the cell-free supernatants frozen. After the last supernatants were collected and frozen, all supernatants were thawed, diluted 1 : 1 with fresh medium, and used as culture medium for freshly NGO-treated LNC or untreated LNC. The results (table 3) show that media taken from NGO-treated and untreated LNC at various times during the j-day period of culture do not enhance the incorporation of [“Hlthymidine by freshly NGO-treated LNC. Likewise, the same media do not augment the incorporation of [3H]thymidine into untreated LNC. Thus, at least in this experimental system, no evidence was obtained for the existence of blastogenic or enhancing factors. Another approach utilized Marbrook vessels for the detection of soluble factors. NGO-treated LNC were cultured on a cellimpermeable membrane (pore size 0.45 pm) in one chamber and the test cells on a similar membrane contained in a second chamber. One chamber fit inside the other in such a way that the membranes supporting the cultured cells were parallel and 3 mm apart. Because the dimensions of the
Table 3. Effect of medium taken from cultures of NGO-treated and untreated node cells on freshly NGO-treated or untreated lymph node cells
rat lymph
Time after start of culture at which the test medium was taken (hours)”
NGO-treated LNC incubated in fresh medium mixed 1 : 1 with test medium taken from cultures of
Untreated LNC incubated in fresh medium mixed I : 1 with test medium taken from cultures of
NGO-treated
2 24 48 72 96 120
172 213 169 180 192 190
LNC
l21+ 7 132* 229f12 349 220& 7 263 189fl 624 319f I 849 579+5 447
Untreated
LNC
NGO-treated
I81 I79 185 198 189 181
789 387 921 176 037 739
I 8912958 2 899t 83 1418?224 2 048k814 1 388+ 57 1468&201
078?~9 683+3 932+6 191+7 278f 1 062f9
LNC
Untreated
LNC
858k I48 I 264f386 I 265f228 I 343*407 1600+716 I 258+165
(’ 2.5~ IO6 NGO-treated or untreated LNC were cultured in 5 ml in a series of 60 mm Petri dishes. At the times indicated above, medium was removed, centrifuged, and the cell-free medium frozen until use. b [3H]Thymidine incorporated during the last 3 h of a 51 h culture period. Average & standard deviation from triplicate cultures.
2 18
Kielian,
Beyer and Bowers
Unlreoted Unlreoted
+NGO-,Mt
C-hofed o
IO 20
so
40
50 60
70
80
go too
6. Abscissa: percent of maximal stimulation. Experiments with Marbrook vessels. Each combination of cells tested is indicated by a roman numeral; the top line gives the treatment of LNC cultured in the inner chamber, the bottom line that of LNC cultured in the middle chamber. [3H]Thymidine incorporated per lo6 cells was highest in all 4 experiments for NGOtreated LNC, and the results of the other combinations were calculated as percent of maximum incorporation. Means and standard deviations are given. The number of experiments performed with each combination is the following: I, 4; II, 4; III, 4; IV, 4; Fig.
v, 2; VI, 4; VII,
2.
membranes in each of the two chambers differed, it was necessary to culture different numbers of cells to achieve approximately equal cell densities. Incorporation of [3H]thymidine during the last 8 h of culture was then determined. The system was checked by adding soluble ConA to one chamber and demonstrating the stimulation of untreated LNC in the adjacent chamber. The normalized results for four experiments are presented in fig. 6. It is clear that untreated LNC were not stimulated as a result of being cultured in proximity to NGO-treated LNC. The percent of maximal [3H]thymidine incorporated by untreated LNC in the test situations (fig. 6, top line of III and bottom line of IV) does not differ from the values obtained with untreated LNC cultured in both chambers (fig. 6, top Exp
Cdl
Rrs
109 (1977)
and bottom lines of I). No diffusable blastogenie factor was therefore detected in these experiments. Another series of experiments was performed in which NGO-treated LNC were cultured in one chamber and a 1 : 1 mixture of untreated LNC and NGO-, mitomycin C-treated LNC in the other chamber. Although the 1 : 1 mixture incorporated more r3H]thymidine than the sum of its components (fig. 6, compare l/2 of the bottom line of I and l/2 of the bottom line of V with the bottom line of VII), as was seen before (fig. 2), there is no indication that NGO-treated LNC cultured in the adjacent chamber influence the response of cells in the 1: 1 mixture (fig. 6, compare the bottom lines of VI and VII).
DISCUSSION Our study deals with two important aspects of NGO stimulation: basic properties of the system and cell interactions leading to lymphocyte transformation. In order to provide a foundation for future investigations with NGO, we have made a detailed study of some of the basic properties of NGO-treated rat LNC. Although stimulation by NGO has been reported for mouse spleen cells [ 11, guinea pig lymph node cells [ 111, and human peripheral blood lymphocytes [12, 131, no comparable study on enzyme conditions, kinetics, or a comparison with other mitogens has been published. The complete kinetic curves presented in fig. 1 indicate that NGO and periodate have similar kinetics. The peak of [3H]thymidine incorporation occurs between 48 and 54 h (figs 1, 2), although NGO treatment results in a greater incorporation of [3H]thymidine than that of periodate, especially at the earlier time points (fig. 1). It seems likely
Cell-cell that more lymphocytes respond to NGO than to periodate, since the sharpness of the peak observed for both periodate- and NGO-treated LNC (fig. 1) suggests that most of the transformed lymphocytes divide only once, as was found for periodatetreated human peripheral blood lymphocytes [14]. Interestingly, the kinetics for ConA-treated LNC follow those of periodate- and NGO-treated LNC during the first 50 h but then rise to a level twice that found for the others before dropping off. Our preliminary autoradiographic data indicate that the frequency of LNC responding to periodate and to ConA treatment during the first 40 h in culture is the same, so it is possible that the progeny of lymphocytes transformed by ConA enter a second division. With regard to the mechanism whereby the action of neuraminidase and galactose oxidase provides a mitogenic stimulus, our results are consistent with the proposal of Novogrodsky & Katchalski [l] that removal of sialic acid by neuraminidase exposes galactosyl residues which are then oxidized by galactose oxidase. Significantly greater transformation results after sequential treatment of rat LNC with neuraminidase and galactose oxidase than after treatment with either enzyme alone or with galactose oxidase followed by neuraminidase (table 1). Simultaneous incubation with neuraminidase (15 U/ml) and galactose oxidase (2.5 U/ml) achieved near-maximal stimulation of rat LNC (table 2) and for convenience was adopted as the standard method of treatment. It is interesting to note that human peripheral blood lymphocytes, in contrast to rat and mouse lymphocytes, show a variable response to galactose oxidase alone [12, 131. The events leading from the initial treatment of LNC with NGO to the eventual blast transformation of large numbers of
interactions
in blast transformation
219
lymphocytes remain to be elucidated. NGO treatment of LNC may lead to transformation in one of three ways: (1) NGO-treated LNC produce factors that lymphocytes require for transformation; (2) an NGOtreated lymphocyte is capable of transforming by itself; (3) cell-cell interactions are required for lymphocyte transformation. Concerning the first possibility, it has been suggested that soluble factors produced by lymphocytes after treatment with mitogens play an important role in blastogenesis [15, 16, 171. In this regard it is advantageous to use NGO treatment as the mitogenic stimulus because the enzymes are removed during the washes prior to culture of the treated LNC and therefore cannot interfere with the detection of any released soluble factors. Despite the potential usefulness of such a system, we were unable to find evidence for such factors in two different experimental systems designed for their detection (table 3 and fig. 6). We conclude that soluble factors do not mediate blast transformation in our system. The possibility that an NGO-treated lymphocyte transforms by itself cannot be excluded, but in fig. 4, NGO-treated LNC diluted with mitomycin C-blocked LNC showed an increase in the [3H]thymidine incorporation per IO6 NGO-treated LNC. If NGO-treated lymphocytes were responding independently of each other and independently of the mitomycin C-treated LNC, then no increase would have been found. Rather, our results favor cell-cell interactions as a requirement for blast transformation induced by NGO treatment. We have exploited the fact that NGO treatment produces a chemical modification on the surface of treated cells to study the kinds of interaction occurring in mixtures of treated and untreated LNC that lead to transformaExp
Cd
Rrs
IOY (IY77)
220
Kielian,
Beyer and Bowers
tion. By this method, we have obtained evidence for two types of cell interaction. One type of interaction, previously termed indirect stimulation [4], can be clearly seen in the situations (figs 2, 3) in which untreated LNC are mixed with NGO-treated LNC rendered incapable of responding by blockage with mitomycin C. The increased incorporation of [3H]thymidine above that of the sum of the components most likely reflects the response of untreated lymphocytes to NGO-treated, mitomycin Cblocked LNC, as was shown previously with periodate by means of karyotype analysis [4]. These results argue for a mechanism of blast transformation that involves an interaction between an NGO-treated stimulator cell and an untreated responder cell. A second type of interaction is suggested by the results of the experiment discussed above (fig. 4) in which NGO-treated LNC were diluted with mitomycin C-blocked LNC. In this case, with increasing dilution the incorporation of [3H]thymidine per lo6 NGO-treated LNC rises to a peak well above the plateau of values obtained for the same NGO-treated LNC diluted with medium. Kinetic differences do not account for these results (fig. 5). Thus it is clear in this situation that mitomycin C-treated LNC assist in the response of the NGOtreated LNC, even though they are incapable of functioning either as stimulators or responders. The relative contribution of these two types of cell interaction to the overall response has not yet been determined. The kinetics found for NGO-treated LNC differ from those obtained with lymphocytes that respond in a purely indirect manner (fig. 2), which suggests that NGO-treated and untreated lymphocytes may respond differently to indirect stimulation by NGO-
treated LNC, or alternatively that mitomycin C blockage may influence the ability of NGO-treated LNC to stimulate. We also found that the magnitude of indirect stimulation depends on the ratio of treated to untreated cells (fig. 3). Nevertheless, it is clear that indirect stimulation contributes significantly to the overall response. In fact, periodate-induced transformation of human peripheral blood lymphocytes has been attributed entirely to indirect stimulation [5; Frost, Monahan & Abell. Personal communication]. The contribution to the overall response resulting from interactions between assisting and responding cells is likewise difficult to assess. The existence of assisting cells is inferred from increased [3H]thymidine incorporation per lo6 NGO-treated LNC when mitomycin C-treated LNC are added (fig. 4), which suggests that assisting cells are present in suboptimal numbers in NGO-treated LNC. Recently it has been found that various lymphoid cell preparations contain vastly different proportions of assisting and responding cells [6]. We have taken advantage of this fact to demonstrate that the addition of preparations containing assisting cells augments the response of periodate-treated lymphocytes up to ISfold [6]. Although it is clear that the cell responding to NGO stimulation is a lymphocyte, the identity of the stimulator and the assisting cell that we have invoked in the two types of cell-cell interactions remains to be elucidated. It is conceivable that the same cell fills both roles. Greneder & Rosenthal [ 11j have shown that there is a macrophage requirement for NGO-induced transformation of guinea pig lymphocytes. In their system, lymphocyte stimulation occurs when purified lymphocytes and macrophages are mixed and either cell type is
Cell-cell NGO-treated. However, some preliminary results argue against an important role for macrophages in our system, but additional work is required before such a conclusion can be reached. Studies on fractionation of rat LNC are currently under way in our laboratory to identify the interacting cells and to determine if macrophages play a role in the response. The authors wish to thank Professor Christian de Duve for many fruitful discussions during the course of this work. This research was supported bv Grants AG00367 and CA-16875 from the HSPHS and Grant IM67 from the American Cancer Society. M. K. is a recipient of a National Science Foundation Predoctoral Fellowship. C. B. is a recipient of USPHS Postdoctoral Fellowship AI-01814. It is also a pleasure to acknowledge the excellent assistance of MS Anne Burdick, MS Barbara Conley, and Mrs Anna Polowetzky.
REFERENCES 1. Novogrodsky, A & Katchalski, E, Proc natl acad sci US 70 (1973) 1824. 2. Spiro, R G, Ann rev biochem 39 (1970) 599. 3. O’Brien, R L & Parker, J W, Cell 7 (1976) 13.
interactions
in blast transformation
221
4. Beyer, C F &Bowers, W E, Proc natl acad sci US 72 (1975) 3590. 5. O’Brien, R L, Parker, J W, Paolilli. P & Steiner, J, J immunol 112 (1974) 1884. 6. Bowers, W E & Beyer, C F, Regulatory mechanisms in lymphocyte activation (ed D 0 Lucas) Academic Press, New York/London (1977) 605. 7. Avigad, G, Amaral, D, Asenio, C & Horecker, B L, J biol them 237 (1962) 2736. 8. Farrar, J J, J immunol I15 (1975) 1295. 9. Hatton, M W C & Regoeczi, E, Biochim biophys acta 438 (1976) 339. IO. Chien. S F, Yevich, S J. Li. S & Li, Y, Biochem biophys res commun 65 (1975) 683. 11. Greineder. D K & Rosenthal, A S, J immunol 115 (1975) 932. 12. Dixon, J F P, Parker, J W & O’Brien, R L, J immunol II6 (1976) 575. 13. Biniaminov, M, Ramat, B, Rosenthal, E & Novogrodsky, A, Clin exp immunol 19 (1975) 93. 14. Norin, A J & Strauss, B S, J immunol I14 (1975) 1683. 15. Novogrodsky, A & Gery, I, J immunol 109 (1972) 1278. 16. Bressler, J, Krzych, U & Thurman, G, Fed proc 35 (1976) 389. 17. Monahan, T M, Frost, A F & Abell, C W. Biothem biophys res commun 73 (1976) 1115. Received March 17, 1977 Accepted April 28, 1977
CELL-CELL OF RAT
Cell Research
109 (1977) 211-221
INTERACTIONS
IN BLAST
LYMPHOCYTES
BY NEURAMINIDASE
GALACTOSE MARGARET Department
C. KIELIAN,
TRANSFORMATION AND
OXIDASE
CARL F. BEYER and WILLIAM
of Biochemical Cytology, The Rockefeller New York, NY 10021, USA
E. BOWERS
University,
SUMMARY Optimal reaction conditions for the in vitro blast transformation of rat lymph node cells (LNC) by neuraminidase (N) and galactose oxidase (GO) were determined. Treatment with either enzyme alone, or with GO before N, did not produce significant stimulation. The kinetics and magnitude of the response to NGO, as measured by [3H]TdR incorporation, were compared with those induced by periodate treatment and by concanavalin A (ConA). Rat LNC treated with NGO (but blocked by mitomycin C) caused blast transformation of untreated syngeneic LNC. The magnitude of this indirect response was about one-quarter of that attained for NGO-treated LNC, and the kinetics were somewhat slower. Supernatants from cultures of NGO-stimulated cells were not able to activate untreated cells, and, using Marbrook culture vessels, no evidence was found for the release of soluble factors that could either activate untreated cells or enhance the magnitude of indirect stimulation. Indirect stimulation, therefore, appears to require cell-cell contact. A second kind of cellular interaction was observed in exueriments in which NGO-treated LNC were cultured at different cell densities in the presence or absence of mitomycin C-blocked filler LNC. In the absence of tiller cells. the incornoration of PHlTdR was a linear function of the number of NGO-treated cells in each culture-over a wide range of cell densities. However, if tiller cells were present, [3H]TdR-incorporation was substantially enhanced. Thus the filler cell population contains cells that assist or potentiate the blast response to NGO, probably by increasing the number of lymphocytes that are stimulated
Novogrodsky & Katchalski first reported that sequential treatment of mouse spleen cells with neuraminidase (N) and galactose oxidase (GO) led to extensive blast transformation [l]. They suggested that removal of the terminal sialic acid residues from cell surface glycoproteins by neuraminidase revealed galactose and N-acetylgalactosamine residues [2], which were oxidized to aldehydes at the C6 positions by galactose oxidase. The function of the aldehyde groups in initiating blast transformation is unknown, but their importance is indicated by the fact that reagents that react with al-
dehydes, such as sodium borohydride or hydroxylamine, can inhibit transformation. An intriguing hypothesis is that the aldehydes form Schiff bases that cross-link molecules on the surface of the same or different cells [ 1, 31. One of the advantages of this mitogenic system is that it allows an evaluation of the role of cellular interactions in the blastogenie response. Since NGO treatment produces a chemical modification of the surface of the treated cells, mixtures of treated and untreated cells can be used to study the importance of cell interactions on the final &I,
Cdl
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109 (1977)
212
Kielian,
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response. Two general classes of interacactivity was measured routinely [7] and was found to be consistently lower by -35% the value spetions between treated and untreated cells cified by the supplier; units in this paper refer to our can be considered: (1) the treated cells in- measured activity. After incubation, LNC were centriand washed once in PBS containing CaCI,. duce transformation of untreated cells, a fuged Periodate. LNC at a concentration of 2X 10’1 ml in process we have termed indirect stimulaice-cold PBS were mixed with an equal volume of 2.4 mM sodium metaperiodate in ice-cold PBS pretion [4]; and (2) the untreated cells influence pared sterilely immediately before use. The suspenthe transformation of the treated cells. sion was kept on ice with occasional gentle agitation for 15 min and then centrifuged at 535 g for 5 min at These interactions could involve cell-cell 4°C. The treated LNC were then washed once in contacts and/or the release of soluble me- HBSS at room temperature. Mitomycin C. LNC were prevented from subdiators. Both kinds of interactions have sequent division by incubating in the dark at 37°C for been observed previously in a mitogenic 30 min IO’ cells/ml in the presence of mitomycin C pg/ml, Sigma, St Louis, MO), in complete mesystem related to NGO, namely the genera- (25 dium consisting of RPM1 1640 (Associated Biomedic tion of cell surface aldehydes by periodate Svstems. Inc.. Buffalo. N.Y.) supplemented with 20% (56°C for 30 minjhorse serum (Grand oxidation of sialic acid residues [4, 5, 61. In heat-inactivated Island Biological Company, Grand Island, N.Y.). The the present study, we have determined the cells were centrifuged and washed once in HBSS. treated with both mitomvcin C and NGO were inreaction conditions for neuraminidase and LNC cubated first with mitomycin C, washed, and then galactose oxidase treatment which lead to treated with NGO. optimal stimulation of rat lymph node cells Cell culture conditions (LNC), and we have compared the kinetics With the exception of the cell culture conditions used and magnitude of the response to NGO for the experiment presented in table I, LNC were with those induced by periodate and by the suspended at a concentration of 3X 106/ml in complete medium. 1.0 ml of a cell suspension was cultured soluble lectin, ConA. We then determined in each well of a Multiwell tissue culture plate (no. the magnitude of indirect stimulation in this 3008, Falcon Plastics, Oxnard, Calif.) incubated at system and investigated whether the cel- 37°C and continuously gassed with a humidified atmosphere of 7 % CO, in air. For each point, triplicate lular interactions involved in this process or quadruplicate cultures were established. Cell viabilities, assessed by trypan blue exclusion, were were mediated by soluble factors. MATERIALS Preparation
AND METHODS
of lymph node cells
Cervical and mesenteric lymph nodes were removed from 150 to 250 g Lewis (LEW) rats (Microbiological Associates, Inc., Bethesda, Md), trimmed free of fat, rinsed three times with Hanks’ balanced salt solution (HBSS), and teased with dissecting needles in sterile HBSS. After rapid filtration through sterile cotton, the lymph node cells (LNC) were centrifuged at 375 g for 10 min and then washed twice in HBSS. Cell counts were made with a Coulter counter, Model B (Coulter Electronics, Inc., Hialeah, Fla).
Treatments and galactose oxidase (NGO). LNC at a concentration of 2xlO’/ml were incubated at 37°C for 30 min in Dulbecco’s phosphate-buffered saline (PBS) containing CaCl, (100 mg/l), 15 U/ml of Vibrio cholera neuraminidase (E.C. 3.2.1.18) (Calbiochem. La Jolla, Calif.) and 2.5 U/ml of Dactyl& dendroides galactose oxidase (E.C. 1.1.3.9) (Worthington Biochemical Corp., Freehold, N.J.). Galactose oxidase Neuraminidase
E.rp Cd
Rrs
determined at the start of the culture period and always exceeded 90 %. In one experiment LNC were incubated in the presence of 25 pg/ml of ConA (MilesYeda, Kankakee, Ill.).
109 (1977)
Labeling of cells with [3H]thymidine, cell harvesting, and preparation for scintillation counting With the exception of the experiments presented in figs 4 and 5, cells were labeled during the terminal 3 h of culture by adding to each well 25 ~1 of a concentrated labeline solution that uroduced a final concentration of 1 X 10mr M thymidine, 300 mCi/mmole of [6-3H]thymidine (SchwarzlMann, Orangeburg, N.Y.: 9 Ci/ mmole). At the end of the ct&ure period, the cells were collected on glass fiber filters (934AH, Reeve Anael. Clifton. N.J.) with the aid of a semi-automatic cell harvester ‘(M12k, Biomedical Research and Development Lab, Rockville, Md). When dry, the filters were placed in scintillation vials, 0.5 ml of Soluene (Packard, Downers Grove, Ill.) was added, and after a minimum of 2 h, 10 ml of a toluene based scintillation fluid containing 22.5 % Triton X-100 and 0.1% acetic acid was added. Radioactivity was measured in a Packard mode1 2002 liquid scintillation spectrometer.
Cell-cell
interactions
in blast transformation
213
Table 1. [3H]Thymidine incorporation by rat lymph node cells treated with neuraminidase and galactose oxidase singly or sequentially [3H]Thymidine
incorporated”
at
1st treatment
2nd treatment
N”
GO’
90 465+2 (100.0)’
GO
N
3 727+74 (3.4)
2 809?63 (2.0)
2 607k581 (2.6)
N
-
983&43 (0.3)
936+336 (0.3)
659294
GO
-
I 355?348 (0.7)
925+59 (0.3)
832YclS3 (0.4)
-
705&239
585+94
4541112
60 h
48 h 757”
72 h
III 901*7 (100.0)
845
84 676&7 (100.0)
205
(0.2)
” During the last 2 h of culture 2 &i of [6iYH]thymidine (9 Ci/mmol) was added to each well of a Multiwell plate initially containing 0.6 ml of LNC (5x lO”/ml) in Dulbecco’s Modified Eagle’s medium supplemented with 20% heat-inactivated horse serum. ’ Fifty units of neuraminidase (N) incubated at 37°C for 30 min with 80x 10” LNC/ml of phosphate-buffered saline (lacking calcium). ’ 0.5 units of galactose oxidase (GO) incubated at room temperature for 30 min with 20x lo6 LNC/ml of phosphate-buffered saline (lacking calcium). ‘r Values given are means f S.D. for triplicate cultures. “ In parenthesis, the percent of [3H]thymidine incorporation relative to that of NGO treatment (100%); background (no treatment) subtracted from each value.
Culture and labeling Marbrook vessels
of cells in
Threaded, periscopic Marbrook vessels (Model B-9008) consisting of an inner, middle, and outer chamber nesting inside of each other were obtained from Bio-Research Glass, Inc. (Vineland, N.J.). Cells were cultured in the inner and middle chambers on membrane filters (Metricil TCM-450, pore size, 0.45 Frn; Gelman Instrument Company, Ann Arbor, Mich.) attached to the chambers by a Teflon O-ring and an annular plastic screw cap. The membrane filter for the inner chamber was 10.6 mm in diameter and for the middle chamber, 22 mm. The Marbrook vessels were prepared for cell culture by a modification of the procedure of Farrar [8]. After a thorough rinse of the chambers with deionized distilled water, chambers were assembled, filled with water, and autoclaved. On the day before use, water was replaced by RPM1 1640 containing 20% heatinactivated horse serum and the vessels were then incubated overnight at 37°C. After removal of the medium, LNC at a concentration of 5~ 106/ml in complete medium were added in a volume of 0.6 ml to the inner chamber and of 2.0 ml to the middle chamber. The outer chamber was filled with 20 ml of complete medium. Marbrook vessels were then incubated at 3PC in a humidified atmosphere of 7% CO, in air. After 44 h, [3H]thymidine was added to all three chambers to bring the radioactivity in each to 0.5 &i/ml, and the vessels were incubated an additional 8 h. At the end of the labeling period, the cells from each chamber were harvested separately onto glass fiber filters (Whatman
GF/C) in a multiple well filtering apparatus (Sampling Manifold no. 1225, Millipore Corporation, Bedford, Mass.). The filters were washed once with ice-cold HBSS, three times with 5 ml of ice-cold 5% trichloroacetic acid, and once with methanol. 0.5 ml of Soluene was added to each filter and radioactivity determined as above.
RESULTS Sequential enzyme treatment is required for LNC stimulation The results presented in table 1 show that extensive stimulation occurred only when rat LNC were treated first with neuraminidase and then with galactose oxidase. Reversal of this sequence or incubation with either enzyme alone was ineffective. Similar results were obtained at all three time points. Determination of optimal enzyme concentrations for LNC stimulation As shown in table 2, treatment of LNC simultaneously with neuraminidase and E.V/> cc// HcsIOY(1977)
2 14
Kielian,
Beyer and Bowers
Table 2. Determination of optimal conditions for simultaneous neuraminidase and galactose oxidase treatment of rat lymph node cells
creasing the galactose oxidase concentration while keeping neuraminidase at a fixed concentration led to a considerably higher degree of stimulation. Less stimulation was noted at concentrations of neuraminidase Neuraminidase Galactose oxidase cont. (U/ml) cont. exceeding 62.5 U/ml and of galactose ox0.1 0.5 2.5 5.0 10.0 idase above 2.5 U/ml, perhaps due to con(U/ml) taminating substances in the enzyme pre2.5 26.1” 52.3 70.1 parations. Commercial galactose oxidase 33.8 56.8 79.0 12.5 and neuraminidase may contain proteases 67.3” 43.4’ [9, lo], and we have observed that Cfo39.5d stridium perfringens neuraminidase (Wor15 91.0 thington) was toxic to LNC at low con62.5 34.9 90.3” 100.0’ 96.2 82.6 centrations. On the basis of these experi97.3’ 100.0” 93.8 72.1 80.9 80.4 ments, standard conditions of 15 U/ml of 82.5 86.1 70.1 Vibrio cholera neuraminidase 125 and 2.5 U/ml of galactose oxidase were chosen for subLNC were treated at 37°C for 30 min simultaneously with neuraminidase and galactose oxidase at the given sequent NGO treatment of LNC.
concentrations. Triplicate or quadruplicate cultures were established for each set of conditions. LNC were incubated for 53 h, labeled with [3H]thymidine, harvested, and prepared for scintillation counting as described in Materials and Methods. a Percent of maximum incorporation of [3H]thymidine. b Simultaneous enzyme treatment for 60 min at 37°C. c Separate enzyme treatment of 30 min each. ’ Lymph node cells treated simultaneously with neuraminidase and galactose oxidase in phosphate-buffered saline lacking calcium. e Results of two experiments.
galactose oxidase was as effective as sequential treatment with the two enzymes. Optimal conditions in this system were established by varying the concentration of enzymes incubated together with LNC at 37°C for 30 min, and the results of two experiments are given in table 2. Since maximal stimulation in both experiments occurred after treatment with 62.5 U/ml of neuraminidase and 2.5 U/ml of galactose oxidase, the other results have been normalized to these conditions. Only a modest increase in stimulation resulted when the galactose oxidase concentration was kept low (0.1 U/ml) and the concentration of neuraminidase was increased, whereas inExp Cdl
Res 109 (1977)
Kinetics of stimulation Aliquots of one preparation of rat LNC were treated with either NGO, periodate, or ConA, and the kinetics of the responses were compared (fig. 1). NGO- and periodate-treated cells showed similar kinetics
0
Fig. 1. Abscissa: time from start of culture (hours); ordinate: [3H]thymidine incorporation (cpmx 10m3). [3H]Thymidine incorporated at various times by LNC that were untreated (O-O) or treated with ConA (CD), NGO (A-A), or sodium periodate (0-O). Each point represents the average of quadruplicate cultures. Standard deviations were less than 13 % in all cases.
Cell-cell
interactions
215
in blast transformation
only half the cells in the mixture can spond, the [3H]thymidine incorporation the peak is about 25 % of that at the peak NGO-treated LNC. A value of 34% is tained by comparing the areas under [3H]thymidine incorporation curves.
reat for obthe
Stimulation at different ratios of NGO-treated to untreated LNC Owing to the important contribution made by indirect stimulation, the composition of 100 40 60 80 0 20 a cell mixture producing maximum [3H]thymidine incorporation was determined. Fig. 2. Abscissu: time from start of culture (hours): ordinafe: [3H]thymidine incorporation (cpmx 10e3). [3H]Thymidine incorporated at various times by un- At constant cell density, the ratio of NGO-, treated LNC (O-O), NGO-treated LNC (O-O), mitomycin C-treated LNC to untreated NGO-treated LNC blocked with mitomycin C (A-A), LNC was varied between the limits of the and a 1 : I mixture of untreated LNC and NGO-treated LNC blocked with mitomycin C (W-B). Each point pure component cell preparations. The represents the average of quadruplicate cultures. averaged results of two experiments, Standard deviations were less than 15% in all cases. presented in fig. 3, show that maximum incorporation of [3H]thymidine occurred in with peak incorporation occurring at 51 h, but the magnitude of the response induced by NGO was significantly greater. In contrast, cells stimulated with ConA showed peak incorporation of thymidine near 70 h, and the magnitude of the response was much greater than for either periodate or NGO. A more detailed kinetic curve for NGO stimulation of LNC is shown in fig. 2, which also includes the kinetics for a 1 : 1 mixture of untreated LNC and NGO-, mitomycin C-treated LNC. Kinetics similar to those already seen in fig. 1 were obtained for NGO-treated LNC, but different kinetics were found for the 1 : 1 mixture. Incorporation of [3H]thymidine peaked between 60 and 66 h and then decreased over the next 2 days. Separate cultures of untreated LNC and LNC doubly treated with NGO and mitomycin C showed at all times a significantly lower [3H]thymidine incorporation than their 1 : 1 mixture. Bearing in mind that
Fig. 3. Abscissa: fraction of untreated cells; ordinate: percent of maximal stimulation. Percent of maximum [3H]thymidine incorporated by mixtures of untreated LNC and NGO-, mitomycin Ctreated LNC which were co-cultured at different ratios but at a constant cell density. [3H]Thymidine was added 3 h before termination of the cultures at 66 h. The background contributed by the untreated LNC and doubly treated LNC to each mixture was subtracted, and the results of two experiments were normalized. Cpm at 100% for the two experiments were 35 000 and 40 000. The means and standard deviations are presented. Eup Cell
Re.7 109 (1977)
216
Kielian,
I@, 0
,
0.3
06
Beyer and Bowers
, 09
,
,
,
I.2
15
I8
, 21
, 24
, 27
, 30
Fig. 4. Abscissa:
responder cell density per culture incorporation (cpm x 10m3)per IO6 responder cells. r3H]‘I;hymidine -incorporated per IO6 NGO-treated LNC (responder cells) by cultures containing different numbers of NGO-treated LNC. Dilutions were made with medium (O-O), mitomycin C-treated LNC (O--O), or NGO-treated LNC blocked with mitomycin C (0-O). Each culture in which NGO-treated LNC were diluted with mitomycin C-treated or NGO-, mitomycin C-treated LNC had a constant cell density of 3 x lo6 cells. r3H]Thymidine was added 4 h before termination of the cultures at 64 h. Standard deviations are indicated in cases where they exceed the size of the symbol.
x 10m6; ordinate: [3H]thymidine
cultures comprised of 70-80% untreated LNC and 20-30 % LNC doubly treated with NGO and mitomycin C.
plete medium gave essentially the same results as found previously for periodate [4]; [3H]thymidine incorporation per lo6 cells was constant between 0.6~ IO” and 3X lo6 LNC/ml, but decreased at cell densities below 0.6~ IO6 LNC/ml. Interestingly, dilution of the same NGO-treated LNC with mitomycin C-treated LNC gave different results. As the proportion of mitomycin Ctreated LNC increased, the incorporation of [3H]thymidine per IO6 NGO-treated LNC also increased until a peak was reached which occurred in cultures containing 0.24~ lo6 NGO-treated LNC and 2.76~ lo6 mitomycin C-treated LNC. Further dilution with mitomycin C-treated LNC reduced the r3H]thymidine incorporation per lo6 NGOtreated LNC. When NGO-treated LNC were diluted with LNC that were treated with both NGO and mitomycin C, the shape of the curve was similar, but an even greater [3H]thymidine incorporation was found. Because the experiment presented in fig. 4 was performed at only one time point, the possibility existed that the differences in [3H]thymidine incorporation for NGOtreated LNC diluted with medium or with mitomycin C-treated LNC actually was due to kinetic differences in the two situations. As shown in fig. 5, the kinetics of proliferating cells at different densities were similar regardless of whether they were diluted with medium or with blocked syngeneic cells. At comparable densities of NGO-treated cells, the incorporation of r3H]thymidine was greater in cultures diluted with mitomycin C-treated cells than with medium.
Stimulation as a function of cell density The possibility that cell interactions may be involved in the response to NGO treatment was further explored by varying the density of NGO-treated LNC. Treated cells were diluted with complete medium or metabolically blocked syngeneic LNC. For the latter, a constant density of 3x IO6 cells per culture was maintained by diluting NGOtreated LNC with either mitomycin Ctreated LNC or LNC doubly treated with Attempt to demonstrate soluble factors in NGO stimulation NGO and mitomycin C. Three experiments yielded similar results; one is presented in The medium from cultures of NGO-treated fig. 4. LNC was tested for the presence of factors Dilution of NGO-treated cells with com- that produce or enhance blastogenesis. A E.rl,
Cdl
RP.\ 109 (1977)
Cell-cell
0
24
48
72
96
120
FI’g. 5. Abscissa: time from the start of culture (hours); ordinate: [3H]thymidine incorporation (cpmx lo-? per culture. Kinetics of [“Hlthymidine incorporation by cultures diluted with medium (O-O) or mitomycin C-treated LNC (O-Cl). Number of NGO-treated cells per culture was: (A) and (B) 1.8x106, (C) and (D) 0.6x10fi, and (E) 0.24~ IO”. Cultures containing NGO-treated LNC and mitomycin C-treated LNC had a constant cell density of 3 x IO@. [3H]Thymidine was added 4 h before termination of the cultures. Standard deviations are indicated in cases where they exceed the size of the symbol.
series of Petri dishes containing NGOtreated LNC or untreated LNC were cultured and at intervals thereafter, medium was taken from each of the two types of cul-
interactions
in blast transformation
217
ture, any contaminating cells removed by centrifugation, and the cell-free supernatants frozen. After the last supernatants were collected and frozen, all supernatants were thawed, diluted 1 : 1 with fresh medium, and used as culture medium for freshly NGO-treated LNC or untreated LNC. The results (table 3) show that media taken from NGO-treated and untreated LNC at various times during the j-day period of culture do not enhance the incorporation of [“Hlthymidine by freshly NGO-treated LNC. Likewise, the same media do not augment the incorporation of [3H]thymidine into untreated LNC. Thus, at least in this experimental system, no evidence was obtained for the existence of blastogenic or enhancing factors. Another approach utilized Marbrook vessels for the detection of soluble factors. NGO-treated LNC were cultured on a cellimpermeable membrane (pore size 0.45 pm) in one chamber and the test cells on a similar membrane contained in a second chamber. One chamber fit inside the other in such a way that the membranes supporting the cultured cells were parallel and 3 mm apart. Because the dimensions of the
Table 3. Effect of medium taken from cultures of NGO-treated and untreated node cells on freshly NGO-treated or untreated lymph node cells
rat lymph
Time after start of culture at which the test medium was taken (hours)”
NGO-treated LNC incubated in fresh medium mixed 1 : 1 with test medium taken from cultures of
Untreated LNC incubated in fresh medium mixed I : 1 with test medium taken from cultures of
NGO-treated
2 24 48 72 96 120
172 213 169 180 192 190
LNC
l21+ 7 132* 229f12 349 220& 7 263 189fl 624 319f I 849 579+5 447
Untreated
LNC
NGO-treated
I81 I79 185 198 189 181
789 387 921 176 037 739
I 8912958 2 899t 83 1418?224 2 048k814 1 388+ 57 1468&201
078?~9 683+3 932+6 191+7 278f 1 062f9
LNC
Untreated
LNC
858k I48 I 264f386 I 265f228 I 343*407 1600+716 I 258+165
(’ 2.5~ IO6 NGO-treated or untreated LNC were cultured in 5 ml in a series of 60 mm Petri dishes. At the times indicated above, medium was removed, centrifuged, and the cell-free medium frozen until use. b [3H]Thymidine incorporated during the last 3 h of a 51 h culture period. Average & standard deviation from triplicate cultures.
2 18
Kielian,
Beyer and Bowers
Unlreoted Unlreoted
+NGO-,Mt
C-hofed o
IO 20
so
40
50 60
70
80
go too
6. Abscissa: percent of maximal stimulation. Experiments with Marbrook vessels. Each combination of cells tested is indicated by a roman numeral; the top line gives the treatment of LNC cultured in the inner chamber, the bottom line that of LNC cultured in the middle chamber. [3H]Thymidine incorporated per lo6 cells was highest in all 4 experiments for NGOtreated LNC, and the results of the other combinations were calculated as percent of maximum incorporation. Means and standard deviations are given. The number of experiments performed with each combination is the following: I, 4; II, 4; III, 4; IV, 4; Fig.
v, 2; VI, 4; VII,
2.
membranes in each of the two chambers differed, it was necessary to culture different numbers of cells to achieve approximately equal cell densities. Incorporation of [3H]thymidine during the last 8 h of culture was then determined. The system was checked by adding soluble ConA to one chamber and demonstrating the stimulation of untreated LNC in the adjacent chamber. The normalized results for four experiments are presented in fig. 6. It is clear that untreated LNC were not stimulated as a result of being cultured in proximity to NGO-treated LNC. The percent of maximal [3H]thymidine incorporated by untreated LNC in the test situations (fig. 6, top line of III and bottom line of IV) does not differ from the values obtained with untreated LNC cultured in both chambers (fig. 6, top Exp
Cdl
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and bottom lines of I). No diffusable blastogenie factor was therefore detected in these experiments. Another series of experiments was performed in which NGO-treated LNC were cultured in one chamber and a 1 : 1 mixture of untreated LNC and NGO-, mitomycin C-treated LNC in the other chamber. Although the 1 : 1 mixture incorporated more r3H]thymidine than the sum of its components (fig. 6, compare l/2 of the bottom line of I and l/2 of the bottom line of V with the bottom line of VII), as was seen before (fig. 2), there is no indication that NGO-treated LNC cultured in the adjacent chamber influence the response of cells in the 1: 1 mixture (fig. 6, compare the bottom lines of VI and VII).
DISCUSSION Our study deals with two important aspects of NGO stimulation: basic properties of the system and cell interactions leading to lymphocyte transformation. In order to provide a foundation for future investigations with NGO, we have made a detailed study of some of the basic properties of NGO-treated rat LNC. Although stimulation by NGO has been reported for mouse spleen cells [ 11, guinea pig lymph node cells [ 111, and human peripheral blood lymphocytes [12, 131, no comparable study on enzyme conditions, kinetics, or a comparison with other mitogens has been published. The complete kinetic curves presented in fig. 1 indicate that NGO and periodate have similar kinetics. The peak of [3H]thymidine incorporation occurs between 48 and 54 h (figs 1, 2), although NGO treatment results in a greater incorporation of [3H]thymidine than that of periodate, especially at the earlier time points (fig. 1). It seems likely
Cell-cell that more lymphocytes respond to NGO than to periodate, since the sharpness of the peak observed for both periodate- and NGO-treated LNC (fig. 1) suggests that most of the transformed lymphocytes divide only once, as was found for periodatetreated human peripheral blood lymphocytes [14]. Interestingly, the kinetics for ConA-treated LNC follow those of periodate- and NGO-treated LNC during the first 50 h but then rise to a level twice that found for the others before dropping off. Our preliminary autoradiographic data indicate that the frequency of LNC responding to periodate and to ConA treatment during the first 40 h in culture is the same, so it is possible that the progeny of lymphocytes transformed by ConA enter a second division. With regard to the mechanism whereby the action of neuraminidase and galactose oxidase provides a mitogenic stimulus, our results are consistent with the proposal of Novogrodsky & Katchalski [l] that removal of sialic acid by neuraminidase exposes galactosyl residues which are then oxidized by galactose oxidase. Significantly greater transformation results after sequential treatment of rat LNC with neuraminidase and galactose oxidase than after treatment with either enzyme alone or with galactose oxidase followed by neuraminidase (table 1). Simultaneous incubation with neuraminidase (15 U/ml) and galactose oxidase (2.5 U/ml) achieved near-maximal stimulation of rat LNC (table 2) and for convenience was adopted as the standard method of treatment. It is interesting to note that human peripheral blood lymphocytes, in contrast to rat and mouse lymphocytes, show a variable response to galactose oxidase alone [12, 131. The events leading from the initial treatment of LNC with NGO to the eventual blast transformation of large numbers of
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lymphocytes remain to be elucidated. NGO treatment of LNC may lead to transformation in one of three ways: (1) NGO-treated LNC produce factors that lymphocytes require for transformation; (2) an NGOtreated lymphocyte is capable of transforming by itself; (3) cell-cell interactions are required for lymphocyte transformation. Concerning the first possibility, it has been suggested that soluble factors produced by lymphocytes after treatment with mitogens play an important role in blastogenesis [15, 16, 171. In this regard it is advantageous to use NGO treatment as the mitogenic stimulus because the enzymes are removed during the washes prior to culture of the treated LNC and therefore cannot interfere with the detection of any released soluble factors. Despite the potential usefulness of such a system, we were unable to find evidence for such factors in two different experimental systems designed for their detection (table 3 and fig. 6). We conclude that soluble factors do not mediate blast transformation in our system. The possibility that an NGO-treated lymphocyte transforms by itself cannot be excluded, but in fig. 4, NGO-treated LNC diluted with mitomycin C-blocked LNC showed an increase in the [3H]thymidine incorporation per IO6 NGO-treated LNC. If NGO-treated lymphocytes were responding independently of each other and independently of the mitomycin C-treated LNC, then no increase would have been found. Rather, our results favor cell-cell interactions as a requirement for blast transformation induced by NGO treatment. We have exploited the fact that NGO treatment produces a chemical modification on the surface of treated cells to study the kinds of interaction occurring in mixtures of treated and untreated LNC that lead to transformaExp
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tion. By this method, we have obtained evidence for two types of cell interaction. One type of interaction, previously termed indirect stimulation [4], can be clearly seen in the situations (figs 2, 3) in which untreated LNC are mixed with NGO-treated LNC rendered incapable of responding by blockage with mitomycin C. The increased incorporation of [3H]thymidine above that of the sum of the components most likely reflects the response of untreated lymphocytes to NGO-treated, mitomycin Cblocked LNC, as was shown previously with periodate by means of karyotype analysis [4]. These results argue for a mechanism of blast transformation that involves an interaction between an NGO-treated stimulator cell and an untreated responder cell. A second type of interaction is suggested by the results of the experiment discussed above (fig. 4) in which NGO-treated LNC were diluted with mitomycin C-blocked LNC. In this case, with increasing dilution the incorporation of [3H]thymidine per lo6 NGO-treated LNC rises to a peak well above the plateau of values obtained for the same NGO-treated LNC diluted with medium. Kinetic differences do not account for these results (fig. 5). Thus it is clear in this situation that mitomycin C-treated LNC assist in the response of the NGOtreated LNC, even though they are incapable of functioning either as stimulators or responders. The relative contribution of these two types of cell interaction to the overall response has not yet been determined. The kinetics found for NGO-treated LNC differ from those obtained with lymphocytes that respond in a purely indirect manner (fig. 2), which suggests that NGO-treated and untreated lymphocytes may respond differently to indirect stimulation by NGO-
treated LNC, or alternatively that mitomycin C blockage may influence the ability of NGO-treated LNC to stimulate. We also found that the magnitude of indirect stimulation depends on the ratio of treated to untreated cells (fig. 3). Nevertheless, it is clear that indirect stimulation contributes significantly to the overall response. In fact, periodate-induced transformation of human peripheral blood lymphocytes has been attributed entirely to indirect stimulation [5; Frost, Monahan & Abell. Personal communication]. The contribution to the overall response resulting from interactions between assisting and responding cells is likewise difficult to assess. The existence of assisting cells is inferred from increased [3H]thymidine incorporation per lo6 NGO-treated LNC when mitomycin C-treated LNC are added (fig. 4), which suggests that assisting cells are present in suboptimal numbers in NGO-treated LNC. Recently it has been found that various lymphoid cell preparations contain vastly different proportions of assisting and responding cells [6]. We have taken advantage of this fact to demonstrate that the addition of preparations containing assisting cells augments the response of periodate-treated lymphocytes up to ISfold [6]. Although it is clear that the cell responding to NGO stimulation is a lymphocyte, the identity of the stimulator and the assisting cell that we have invoked in the two types of cell-cell interactions remains to be elucidated. It is conceivable that the same cell fills both roles. Greneder & Rosenthal [ 11j have shown that there is a macrophage requirement for NGO-induced transformation of guinea pig lymphocytes. In their system, lymphocyte stimulation occurs when purified lymphocytes and macrophages are mixed and either cell type is
Cell-cell NGO-treated. However, some preliminary results argue against an important role for macrophages in our system, but additional work is required before such a conclusion can be reached. Studies on fractionation of rat LNC are currently under way in our laboratory to identify the interacting cells and to determine if macrophages play a role in the response. The authors wish to thank Professor Christian de Duve for many fruitful discussions during the course of this work. This research was supported bv Grants AG00367 and CA-16875 from the HSPHS and Grant IM67 from the American Cancer Society. M. K. is a recipient of a National Science Foundation Predoctoral Fellowship. C. B. is a recipient of USPHS Postdoctoral Fellowship AI-01814. It is also a pleasure to acknowledge the excellent assistance of MS Anne Burdick, MS Barbara Conley, and Mrs Anna Polowetzky.
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4. Beyer, C F &Bowers, W E, Proc natl acad sci US 72 (1975) 3590. 5. O’Brien, R L, Parker, J W, Paolilli. P & Steiner, J, J immunol 112 (1974) 1884. 6. Bowers, W E & Beyer, C F, Regulatory mechanisms in lymphocyte activation (ed D 0 Lucas) Academic Press, New York/London (1977) 605. 7. Avigad, G, Amaral, D, Asenio, C & Horecker, B L, J biol them 237 (1962) 2736. 8. Farrar, J J, J immunol I15 (1975) 1295. 9. Hatton, M W C & Regoeczi, E, Biochim biophys acta 438 (1976) 339. IO. Chien. S F, Yevich, S J. Li. S & Li, Y, Biochem biophys res commun 65 (1975) 683. 11. Greineder. D K & Rosenthal, A S, J immunol 115 (1975) 932. 12. Dixon, J F P, Parker, J W & O’Brien, R L, J immunol II6 (1976) 575. 13. Biniaminov, M, Ramat, B, Rosenthal, E & Novogrodsky, A, Clin exp immunol 19 (1975) 93. 14. Norin, A J & Strauss, B S, J immunol I14 (1975) 1683. 15. Novogrodsky, A & Gery, I, J immunol 109 (1972) 1278. 16. Bressler, J, Krzych, U & Thurman, G, Fed proc 35 (1976) 389. 17. Monahan, T M, Frost, A F & Abell, C W. Biothem biophys res commun 73 (1976) 1115. Received March 17, 1977 Accepted April 28, 1977
