Activation of lymphocytes by concanavalin a requires calcium ions

Cell Biology

international

ACTIVATIONOF

Reports,

LYMPHOCNES

Vol. 3, iw.

35

1, 1979

BYCONCANAWLINARFQUIRRSCALCIUM

SusanM.Milner,Departrwntof Tennis Court Road, Cambridge,

IONS

Pathology, England

ABSTRACT

Pbuse spleen cells were exposed to a short pulse of the mitogenie lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine intoDNA. It was found: (a) the nwber of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitcyenic effect an extracellular calcium cancentration>10-5M was required during the time that the cells were exposed to con A. INTRODU'2l?IoN Themitogenic property of Ca++ ionophores suggests that an increase in cytoplasnic calcium levels will activate resting cells frcxn Go into Gl of the division cycle. In sane instances the calcium ionophore is likely to be a primary mitogen, for example, in the activation of unfertilised eggs by ionophore A23187 (Steinhardt etg., 1974). Furthermore, these wxkers found that redistributiZ of intracellular calcium may serve as the source of calcium for the increment of cytoplasmic calcium levels, since A 23187 will activate unfertilised eggs in the absence of extracellular calcium. In nollticellular systems such as lymphoid tissue, it is kncwn that various cell types contribute towards the net cellular response to mitogen. In addition to the various interactions between different T cell subpopulations, B cells and monocytes, (Arala-Chaves et al., 1978), Habu and Raff (1977) found that at least sane muse lymphocytes have an absolute requirmt for macrophages in their mitogenic response to con A. A key question in cell growbh control is whether an increase in cytoplasmic calcium acts as a universal means of initiating cell division. If thiswere true onewould expactmitogenic plant lectins to alter the levels of calcium in the ell cytoplasm. Attempts to measure directly the uptake of Ca 45 into lymphocytes on exposure to various T cell mitqens have led to conflicting results. Several wrkers report an increased influx of Ca45 into lymphocytes 0309.1651/79/010035-09/$02.00/O

01979

Academic

Press

Inc.

(London)

Ltd.

36

Cell Biology

International

Reports,

Vol. 3, No. 1, 1979

withinlhexpxuretomitcgen, although their results vary widely in detail (Allwood et &., 1971; Whitney and Sutherland, 1973; Parker, 1974; Hoviyt al., 1976; F&&man et al., 1975). Other workers (Hesketh et al., 1977) failed to detect a change in the Ca45 influx into lymphccytes qsed to mitogenic concentrations of con A or A23187. The experimental problems of accurately measuring an increased Ca45 influx against a high background, togetherwith the differat assay techniques employed by the different groups, may partly account for the confusion in this field. In view of the experimental difficulties of directly measuring and interpreting Ca45 fluxes, I have adopted. an alternative approach: namely to define the requirements for extracellular calcium during the mitogenic interaction of con A with lymphocytes. Cells are activated by brief exposure to con A. The concentration of extracellular calcium was varied during the con A treatment, afterwhich conAwas removed and the cells incubated inmedium containing calcium. Despite reports to the contrary, I have found that a significant nti of lymphccytes became ccxrmitted to enter the division cycle after brief exposure to con A (Milner, 1977, and This is in agreement with Gunther --et al. (1974) who this paper). showd thatsanelymphocytes becane ccxrmittedtoDNA synthesis as early as 2 h after exposure to con A and that the rxxnber of cells induced into mitosis by con A increased with increasing time of exposure to the lectin until after 20 h when the response was maximal. They suggest that different cells differ intrinsically in the time of exposure to lectin necessary to cannit them to DNA synthesis; alternately it is argued that cells in the non-dividing state, Go, may cycle through various metabolic states, only scme of which are sensitive to mitogenic stimulation out of Go into G1 (also see Temin, 1972). This mitogen sensitive subpopulation would contain those cells able to be comnitted to cell division by brief exposure to con A.

The basic experimental protocol is described in fig. variations frcxn this format are described in the relevant Experimental con A pulse

1 and legends.

Prccedure

removal of bxndconA

Incubate to measure induced mitogenesis

Control

i-d-+

-Ca++

+ca*

Ekperiltlental

-Ca++

-ca++

tCa*

2h

2h

68h

Figure 1: Experimental design for exposing pulse of con A. Spleen cells from a single

spleen cells to a short Balb/c mouse (6-10 weeks

Cell Biology

International

Reports,

Vol. 3, No. 1, 1979

37

<,ld) were washed with 0.2 mM EGTA in MEM for suspension culture (Flow Laboratories, containing approximately 3 x 10e6M calcium as The I)-Ca pantothenate; hereafter referred to as Ca++ -free MEM). washed cells were spun down and sepa-rate aliquots were resuspended to a final concentration of 5 x LO6 (cells ml-l in either Ca++ -free> MEM, or in MEM supplemented with 0.2 mM CaC12. Con A (Miles-Yeda; IO0 .~g ml-1 stock solution in Ca ++ -free MEM) was added to each c.ei ,;uspension to give a final concentration of ip g or 5 yg ml-l and iive replicate cultures (10O)~l) of each suspension dispensed l~ntct I,inbro microculture plates at time zero. The cultures were incubateti for 2 h at 37OC in a sealed, humidified box gassed with 10". ?02, 72, 02 and 83% N2. Unbound con A was removed from all culture:; by complete exchange of the supernatant for Ca++ -free MEM cL>n-Laininq 100 mM a-methyl mannoside (W-MM). Cell-bound con A was ,!isp!aced by the cr-MM during a further 2 h incubation after which he supernatant was exchanged a secc'nd time and all cultures were lmed identically with RPM1 1640. Incubation was then continued for 1;; 1, and tritiated thymidine was added at 48 h (0.5~ c per culture, Thus, the only difference? in spec-ific activity 2.0 Ci mmole-1). rreatment of the cultures was in the first 2 h incubation step. In preliminary experiments 0.2 mM EGTA was included in the Cat+ -free the presence of EGTA did riot -ilter MEM during the 2 h con A pulse; the results compared to those from cells pulsed in Ca'+ -free MEM dlor:e, and any residual Ca++ in Ca+- -free MEM was discounted forthe purpose of these experiments. Although 1 ~19 ml -1 con A ‘diiS fouI(d to be optimal mitogenic concentration during continuous exposure to the lectin in these conditions, relatively high con A cont.entrations were found necessary for pulse activation. Roth ME.M and RPM1 rr;;;:,;edsd;,w;;; ,ssr;;I:ree, the only additions being 100 u ml.=treptomycin and 1% glutamlnr. The Mg++ content of MEM and RPM1 1640 is 10V3 M and 4 x 10e4 M rpy:-pectively. RPM1 contained 0.43 mM 'Ca ++ .

Cultures exposed to a 2 h pulse of con A in the presence of 0.2 Ml CaC12 contained a significant proportion of cells which underwent mitogenesis (fig. 2). In contrast, cells treated identically, except for the absence of extracellular calcium during exposure to con A, failed to enter S phase of the division cycle within 72 h although Mg* was present throughout and 0.43 n-&l Ca* was added after removal of lectin, i.e. during the final 68 h of culture. Control experiments showed that the failure of spleen cells to respond to con A in the absence of calcium was not due to impairment of con A binding to the cell surface (fig. 3a); nor to any toxic effects, due, for example, to culturing cells for 4 hours in the absence of calcium (fig. 3b). These experiments shm that mitogenesis by con A has an absolute requirement for extracellular calcium. Furthemre, cells incubated with Ca* -free MEM containing 0.2 rrN EGTA for 1 h imnediately before, or after, pulsing with con A plus 0.2 rrM CaC12 responded to the con A pulse to the sama extent as did cells cultured in the continuous presence of calcium. Thus the lack of extracellular calcium blocked the generation of a

Cell Biology International

38

Reports, Vol. 3, No. I, 1979

20

0

I llil

background ho con A)

Figure 2: Response of spleen cells to a con A pulse in Ca++ -free medium. Spleen cells were pulsed with 3 or 5 pq ml-' tori A in Ca++ -free'MEM, or in MEM supplemented with 0.2 mM CaCl' as described in the legend to fig. 1. This type of experiment was repeated eight times and, although there was some variation in the amplitude of the response of cells from different spleens pulsed in the presence of Ca++ I cells from the same spleen pulsed in the absence of Ca++always showed between 90-100% inhibition of mitogenesis. I represent cultures pulsed in the presence of 0.2mM CaC12 andm represent cultures pulsed in Ca++ -free MEM. Tritiated thymidine incorporation in cultures pulsed with 3 ug and 5 ug ml-l con A plus Caf+ were 3505 (* 434) cpm and 16420 (' 3211) cpm respectively. Binding and toxicity controls are shown (for details see fig. 3).

c. p. m Vitiated

thymidine

incorporation

(x 10

-3 )

CR -.

40

Figure

Cell Biology

International

Reports,

Vol. 3, NO. I, 1979

Toxicity of experimental treatment.

3b:

100

v)

80

2c 0 Control

Experimental

Figure 3a: Thebinding of con A to spleen cells in the presence or absence of extracellular Ca++ ions. Spleen cells were suspended in Ca++ -free MEM or in MEM supplemented with 0.2mM CaC12 as described in fig. 1. Various amounts of con A were added to aliquots of each cell suspension and five replicate cultures of 100 ~1 in Linbro microculture plates were incubated for 1 h. Unbound con A was then removed from the cultures by complete replacement of the supernatant with prewarmed RPM1 1640. The cells, together with any cell-bound con A, were then incubated for 48 h and labelled with tritiated thymidine for a further 3 h. The con A dose-response curve is shown for cells which bound con A in the presence of 0.2 mM CaC12, 0; or in Ca++ -free MEM, I. Figure 3b: Mitogenic responsiveness of spleen cells pulsed with con A in the presence or absence of extracellular Ca++ ions. The method of pulse activation is described in fig. 1. In this control 62 h incubation experiment 1 I-lg ml-l con A was included in the final step in RPMI. DNA synthesis was measured between 48-72 h. Repeated experiments showed a 5-102 reduction in the mitogenic responsiveness of cells pulsed with con A in the absence of extracellular Ca++. The response of cells to lug ml-l con A after pulsing in 0.2 mM CaC12 or in Ca++-free MEM is shown by aand mrespectively.

Cell Biology

International

Both binding L;ince mouse

Reports,

41

Vol. 3, No. 1, 1979

and toxicity controls were to mouse variation occurred.

included

in

all

experiment:;

mitcqenic respoke during theperiodt.hatconAwaskmndto the mriments cell surface, rather than at closely related times. designed to find the limiting concentration of extracellular calcium required for con A showed that levels abve lo-5M were required during the 2 hpulse. The response was optimal at 5 x 10m4M CaC12 (fig. 4).100

80

.-m xi

60

I 0 0

up

1o-5

1o-4

lo+

M CaC12 Figure 4: CA++ requirement for spleen cell mitogenesis during pulse _l__ activation with con A. Aliquots of spleen ceils were suspended ;n MESI supplemented with CaC12 concentrations ranging from lT6 tn 10e3 M and pulsed with 3 pg ml-l con A as described in fig. 1. +ve replicate cultures were used for e?ch point. In some cases DNA synthesis was measured between 24-72 h to avoid any discrepancl!

42

cell Biology

International

Reporn,

Vol. 3, No. 1, 1979

between cultures due to a possible effect of Ca++ on the kinetics of the cell response to con A; the results were the same as shown here. In each of the 3 experiments shown, some cells were also cultured in the continuous presence of 1 ug ml-l con A plus various CaC12 concentrations in MEM for 72 h, and DNA synthesis was measured between 48-72 h. The requirement for calcium in this chronic experiment was the same as for pulse activation. The proportion of cells stimulated into mitosis during the 2 h pulse was calculated in terms of the total cell population able to respond to con A, i.e. those cultured in the continuous presence of con A. The maximum cpm incorporated in the pulse and chronic experiments were 2053 ('455) and 12,000 ('4623) ino; 9820 ('3097) and 130,000 ('3832) in A; and 31,000 ('3637) and 171,000 ('10,331) inxrespectively. Since maximal commitment of the cells to DNA synthesis occurs at about 20 h, the proportion of cells responding to a 2 h pulse of con A from the 3 different mice ranged between 6% and 18% of the total responsive population.

DISCUSSION Fulse activation experiments enable analysis of the conditions required for the triggering of mitogenesis to be separated from subsequent events. Since relatively high concentrations of con A are necessary for pulse activation it might be argued that the treatmentwith a-MMonlypartlyremoved the conA, leaving behind sufficient residual con A to induce mitogenesis during the incubation in RPNt. Control experiments shaw that any residual lectin bound onto cells in the absence of Ca* failed to initiate mitogenesis in the final 68 h of culture, although the potential of these cells for stimulation by con A had not been impaired (fig 3b). As the amount of conAbxndduring the 2 hpulseis not influenced by the presence of Ca* in the medium, this argues very strongly that triggering of mitogenesis occured in the first 2 h of culture when the cells were plsed with con A. In agreement with this is the fact that the proportion of cells responding to a 2 h pulse of con A ranged between 6% and 18% of the total population able to respond to con A (measured as the response in the continuous presence of con A). These results are in agreement with Gunther --et al. (1974) in that the number of cells responding to con A is proportionalto thetimeof their exposure to thelectin (seelegendto fig. 4). In conclusion it may be stated that extracellular calcium ions are required for the mitogenic action of con A on mouse spleen cells, although whether the calcium acts as a direct mitogen, or functions to release mitcgenic factor(s), has yet to be determined.

This mrk was supported by a grant frcxn the Medical Research Council.

Cell Biology

International

Reports,

43

Vol. 3, No. 1, 1979

REFTBENCES Arala-Chaves, M.P., Role of adherent and concanavalin

Hope, L., Kom, ,J.H. and E'udenberg, A. (1978). cells in immune responses to phytohaw&agglutinin A. European Journal of Imnunology 8, 77-81

G.L., Davey, ?l.J. and Goodford, P.J. (1971) m Alhod, G., Asherton, The early uptake of radioactive calcium by human lymphocytes treated with phytohaemagglutinin. Imnunolcgy 21, 509-516 Freedman, M.H., Raff, M.C. and Gcxnperts, B. (1975). Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides. Nature 255, 378-382 Gunther, G.R., Wang, J.L. and Edelman, G.M. (1974). The kinetics of cellular oxnnimt during stimulation of lymphocytes by 1ectins. Journal of Cell Biology 62, 366-377 Hah,

S. and Raff, M.C. (1977). Accessory cell dependence of lectin-induced proliferation of mouse T lymphocytes. European Journal of Immunology 7, 451-457

Hesketh, T.R., Smith, G.A., Houslay, Is an early calcium J.C. (1977). lymphocytes? Nature 267, 490-494

M.D., Warren, G.B. ax-d Metcalfe flux necessary to stimulate

Hovi, T., Allison, A.C. and Williams, S.C. (1976). Proliferation of human peripheral blood lymphocytes induced by A23187, a Streptcqces antibiotic. Experimental Cell Research 97, 92-100 Milner, S.M. (1977). Activation of mouse spleen short pulse of mitcgen. Nature 268, 441-442

cells

by a single

Parker, C.W. (1974). Correlation between mitogenicity and stimulation of calcium uptake in human lymphocytes. Biochemical and Biophysical Research Cm ications 61, 1180-1186 Steinhardt, R.A., Epel, D., Carroll, Is calcium ionophore a universal eggs? Nature 252, 41-43 Temh, H.M. stationary

E.J. and Yanagimashi, R. activator for unfertilised

(1972). Stimulation by serum of mltiplication chicken cells. Journal of Cellular Physiology

Whitney, R.B. and Sutherland, R.M. (1972). Enhanced uptake calcium by transforming lymphocytes. Cellular Immunology 137-147

Received:

7th

August

1978

Accepted:

21st

August

(1974).

of 78,

161

of 5,

1978