Lectin-induced lymphocyte agglutination

Printed in Sweden Copyright Q 1973 by Academic Press. Inc. A// righfs oJ raproducfion in any form resewed

Experimental Cell Research 82 (1973) 415-425

LECTIN-INDUCED

LYMPHOCYTE

An Active Cellular

AGGLUTINATION

Process?

FRANCIS LOOR Basel Institute for Immunology,

CH-4058 Basel, Switzerlund

SUMMARY The agglutination of lymphocytes by lectins (Concanavalin A, phytohemagglutinin and pokeweed mitogen) is studied with fluorochrome-labelled lectins and by fluorescence microscopy. Both agglutinated and non-agglutinated cells pick up similar amounts of le.&ins as judged by their equivalence cell membrane brightness. At the level of resolution of optical microscopy no difference can be detected in the distribution of lectins bound by agglutinated or non-agglutinated cells. However a profound inhibition of agglutination by inhibitors of the capping process (sodium azide and cytochalasin B) suggests the involvement in the agglutination process of a step that requires active cell metabolism and microfilament function.

Lymphocytes can be readily agglutinated by the lymphocyte membrane in relation to their lectins. These plant agglutinins are proteins mitogenicity [4], we have obtained evidence with anti-carbohydrate specificities, found that this passive agglutination hypothesis is to be mostly di- or tetravalent and reported not fully satisfactory, and that agglutination to be of low affinity for their known ligand involves an active participation of the agglu(reviewed in [l]). Their binding sites on the tinated cells themselves requiring microfilacell membrane are presumably located in ment function. the glycocalix which is constituted by sugar moieties of plasma membrane glycoproteins MATERIALS AND METHODS and glycolipids [2]. Cells presenting a clustering of the lectin- Cells. Thymus (NT), spleen (NS), inguinal and mebinding sites are more readily agglutinable senteric lymph nodes (NLN) from BALB/c mice used as a source of lymphocytes. In some exthan when binding sites are evenly distri- were oeriments we used thoracic duct lymphocytes from buted; moreover conditions known to favour CBA mice either normal (NTDL) or activated against histocomnatibilitv antigens (TTDL) 151. membrane component redistribution, mainly C57Bl16 NTDL and TTDL were - kindl; provided by ‘Dr higher temperature, favour the agglutina- Jonathan Sprent. suspensions were prepared according to Pertion. These findings have been interpreted nisCell et al. [6]. Lectins. They were purified and conjugated with as supporting the hypothesis that the agTetramethyl Rhodamine isothiocyanate glutination was due to the cross-linking of either (TRITC) or fluoresceine isothiocyanate (FITC). Concanavalin A (ConA). Con A purchased from the cells by the plant lectins [3]. Miles-Yeda or from Sigma was further purified by During the course of a study on the re- affinity chromatography on Sephadex G 50 [7]. The distribution of agglutinin binding sites on purified Con A was conjugated with either FITC 28-

731808

Exptl Cell Res 82 (1973)

416 F. Loor

20

30

40

50

60

70

Fig. 1. Abscissa: tube no.; ordinate: absorbance at 280 nm, 515 nm. o---o, Azso; o-o, A,,,. Affinity chromatography of TRITC-Con A on a Sephadex G 50 column in PBS, pH 7.5. Arrow indicates time of addition of 0.1 M glucose in PBS.

(12.5 ,ng/mg protein) or TRITC (30 ,eg/mg protein) for 1 h at 0°C at pH 9.0 and in the presence of 0.1 M glucose in order to protect the active sites. The sugar and the excess fluorochrome were dialysed by continuous dialysis. The fluorescent Con A was then separated by affinity chromatography on Sephadex G 50, the fully active ConA being eluted by 0.1 M glucose (fig. I). After extensive continuous dialysis to eliminate the glucose, the fluorochrome-conjugated ConA preparations were kept either lyophilized or in concentrated protein solution, in saline containing 1 mM MnCl, and 1 mM CaCl,, without any loss of activity for weeks at +4”C. Two fluorescent ConA preparations have been essentially used: FITC Con A with a ratio OD 280 nm/OD 495 nm of 1.0, and TRITC ConA with a ratio OD 280 nm/OD 515 nm of 3.3. Amounts of ConA engaged in the experiments were calculated on the basis of Ei $, = 11.4 at 280 nm. Phvtohemaanlutinin (PHA). PHA-P vurchased from-DIFCGwas further purified on a column of hvdroxvlavatite cellulose (SERVA). Peaks of material eiuted by phosphate‘buffer pH 7.5, 0.005 M and 0.05M were discarded and only the peak of protein eluted by the buffer 0.5 M was kept as ‘pure PHA’ [8] (fig. 2). This pure PHA was conjugated either with FITC or with TRITC by the same general technique described for ConA. No sugar was added to protect the binding site. Excess of free fluorochrome was eliminated by passage on Sephadex G 50 and extensive dialysis. Two fluorescent PHA preparations have been essentially used: FITC PHA with a ratio OD 280 nm/OD 495 nm of 2.05 and TRITC, PHA with a ratio GD 280 nm/OD 515 nm of 3.4. Amounts of PHA engaged in the experiments were calculated on the basis of Ei 2m = 12.0 at 280 nm Pokeweed mitogen (PWM). PWM purchased from GIBCo was further purified on a column of hydroxylExptl Cell Res 82 (1973)

apatite cellulose (SERVA) [9]. This fractionation is shown in fig. 3. Three peaks of material were obtained by stepwise elution with phosphate buffers pH 7.5, 0.005, 0.05 and 0.5 M. The first peak contained essentially dialysable material (passing through Diaflo membrane UM 2) and was discarded. The third peak consisted of a protein similar to PHA by Agar electrophoresis at pH 8.6 (cathodal migration) and was also discarded. The second peak contained a protein with anodal migration in the agar electrophoresis and was considered as ‘pure PWM’ [9]. PWM was conjugated with TRITC in very mild conditions: a solution of pure PWM at 10 mg/ml in saline brought to pH 8.5 with NaOH was dialysed at 0°C overnight against 10 times its volume of TRITC (300 ,ug/mg protein) dissolved in the same medium. Unfixed fluorochrome was then removed by passage on Sephadex G 50 column followed by extensive dialysis. The TRITC-PWM used in these experiments had a ratio of OD 280 nm/OD 515 nm of 4.5. The amounts of PWM engaged in the experiments were calculated on the basis of Ei & = 14.5 at 280 nm. Agglutination assay. The cells were suspended in Dulbecco PBS (Ca2+ and Mg2+ free) pH 7.2, supplemented with 0.5 % BSA. Medium with foetal calf serum usually engaged for checking the mitogenic activity of these lectins was avoided in the exneriments to be described here, because the background agglutination was very variable and often high,

0.5M

I

y 30

40

50

A

60

\

,J--,

70

60

,h

90

Fig. 2. Abscissa: tube no.; ordinate:

100

110

120

130

A,,,. Purification of PHA. 100 mg of PHA-P (GIBCo) was applied on a column of hydroxylapatite cellulose (Serva) 20 cm high x 2.5 cm 0 in phosphate buffer, pH 7.5, 0.005 M. Arrows indicate times of addition of 0.05 and 0.5 M buffers respectively for elution of second and third peaks. Peak III is PHA.

Lectin-induced lymphocyte agglutination 4 17

Fig. 3. Abscissa: tube no.; ordinate: A,,,. Purification of PWM. Forty mg of PWM (Difco) was applied on a column of hydroxylapatite cellulose 30 cm high x 0.9 cm 0 in phosphate buffer, pH 7.5, 0.005 M. Arrows indicate times of addition of 0.05 M and 0.5 M buffers respectively for elution of second and third peaks. Peak II is PWM.

depending on the particular batch of foetal calf serum available. According to our observations on the inhibition of the capoina _. -_urocess 141. _-_ ‘cap’ . inhibitors included in the BSA-PBS were either 1O-2 M sodium azide (NaN,) or 10e4 M Cvtochalasin B (CB) in 0.5 ‘% DMSo (dimethylsulfoxide). Cytochalasin R was obtained from ICI Research Laboratories. To investigate if cap inhibitors did inhibit lectin-

induced agglutination (‘inhibition assay’), 2.5 ’ IO” lymphocytes were incubated in 10 mm diameter centrifuge tubes for 30 min at 37°C in 1.0 ml medium with or without cap inhibitor, then spun (300 g, 15 min) and resuspended in 0.1 ml of their respective media. The fluorescent lectin was then added and the cells incubated for 30 min at 37’C with occasional gentle shaking or rotating by hand, washed once, resuspended in 50-100 ,ul medium and examined microscopically in suspension. In attempts to revert the lectin induced agglutination by cap inhibitors (‘reversal assay’), cells were first agglutinated by lectin for 30 min at 37’C in absence of any inhibitor, washed once, suspended in 1.0 ml medium with or without NaN, or CB, incubated with gentle shaking for 30 min at 37 C. spun down and finally observed microscopically in suspension as above. Microscopy. The cells were first examined at a magnification of 125 to 500 to evaluate the degree of agglutination (scored as number of 1 , fig. 4) and then at I 250 magnification to study the distribution of bound fluorescent agglutinin by fluore-scence microscopy [6, lo] (Leitz Orthoplan microSCOD~ eauinoed with Osram HBO-200 mercurv vanour- lamp and Opak-Fluor vertical illuminator): ’ In order to get a more quantitative view, in some experiments, the relative percentages of ‘free’ and ‘clumped’ cells were counted using the higher msg. nification. At least 5 000 cells were counted for each sample and the values obtained for different assays with a given lymphocyte preparation were remarkably similar. Slight variations (less than 20”,.) occurred from one mouse to another.

Fig. 4. Scoring the degree of lymphocyte agglutination of PHA (from the experiments reported in table I). (a) -, complete inhibition of lymph node cell agglutination by lo-” M CB; (b) , partial agglutination of A I ) complete agglutiration of lymph node cells (conlymph node cells in presence of 10 mM NaN,; (c) trol DMSO). Phase contrast. Exptl Cell Res 82 C/973)

418 F. Loor

Fig. 5. Binding of fluorochrome-conjugated PWM to thymus cells. (a) fluorescent staining of dissociated cells (in presence of CB); (b) same cells as (a), in phase contrast; (c) fluorescent staining of clustered cells (no ‘cap’ inhibitor present); (d) same cells as (c), in phase contrast. Not all the cells of the cluster are in focus, which explains apparent differences of brightness.

RESULTS Results essentially relevant to the problem of redistribution of agglutinin-binding sites on the lymphocyte membrane, to the free mobility of other membrane components on lectin coated lymphocytes and to the mitogenicity of our fluorochrome-labelled lectins are described and discussed in detail in other papers [4, II]. Unless otherwise stated, the fluorescent lectins were used at physiological concentration, i.e. at doses at which the other studies [4, II] showed that those lectins could be easily redistributed on the lymphocyte membrane, and did not reduce the free mobility of membrane immunoglobulins and showed an optimal mitogenicity for normal spleen Exptl Cdl Res 82 (1973)

or lymph node lymphocytes. Then we have used 2.5 ,ug/ml ConA, 25 pug/ml PHA and 50 pug/ml PWM. PHA-induced agglutination The observation relevant to the mechanism of cell agglutination was made during studies on PHA redistribution on lymphocytes. In ‘capping’ conditions (37°C no metabolic inhibitors), it appeared that lymphocytes were agglutinated. In ‘non-capping’ conditions (presence of NaN, or of CB), there was a striking reduction in the degree of agglutination of the cells. This point was further investigated by trying (a) to inhibit and (b) to revert agglutination. As shown in table 1, CB very efficiently inhibited the PHA induced agglutination of

Lectin-induced lymphocyte agglutination

419

Table 1. PHA-induced lymphocyte agglutination Lymphocyte source

Sample Assay

PHA

I Inhibition (I)

Reversion (2)

1

Medium Control Control Control DMSO (3) + lob2 M NaN, -lo-* M CB Control Control Control DMSO A 1O-2 M NaN, i-10+ M CB

-NT

N LN

(18) (944) (91,

(14)

’ !

! ,!

I

(80)

(794) (494) (8B) (N.C.) (781) (786) (49) (34)

’

(92)

(394) (N.C.) L (95) -’ (954) (94) 2 1 (93)

PHA at 25 pug/ml. N LN, normal lymph node lymphocytes; NT, normal thymus lymphocytes; N.C., not counted. (1) First medium + inhibitor, then agglutinin; (2) first agglutinin, then medium + inhibitor: (3) control medium with 0.5 “4,DMSO. Numbers within parentheses represent percentages of clumped cells, counted in 1 expt.

lymph node lymphocytes and significantly reduced the agglutination of thymus lymphocytes. NaN, could reduce the agglutinability of lymph node lymphocytes but not that of thymocytes. Thymocytes were always found in all our assays to be clearly more easily agglutinable. As described in other studies [4], the fluorescent PHA, initially bound as a ring on the lymphocyte membrane, was partially redistributed into patches on presence of cap inhibitors; when no cap inhibitor was present, it was partially capped (some was left as a faint ring on the membrane). The capping was faster on isolated cells than on cells in the agglutinates [4]. When observed before the capping process could start, i.e. immediately after a treatment of the cells at 0°C and with an equipment allowing the observation of the cells at O4°C [IO], there was no detectable difference in the amounts of PHA bound by the cells, whether they were in agglutinates or not. This was judged by the equivalent brightness of their cell membrane. When the capping of PHA was inhibited by the low temperature both during incubation of the cells with PHA and during microscopic observation,

again no detectable difference could be found for the amounts of membrane bound fluorescence, whether a cap inhibitor was present or not. The only difference was related to the cell origin, as thymocytes bound significantly more PHA than lymphocytes from other sources [4]. In reversal attempts, only agglutinated lymph node lymphocytes could be efficiently dissociated into free lymphocytes by the CB or partially dissociated by NaN,, while the thymocytes were not. After dissociat i of the agglutinated cells, the membrane fluorescencewas distributed as patches on a diffuse ring background, as it was the case for cells treated in presence of cap inhibitors [4]. P W&f-induced agglutination

While less extensively studied so far, PWMinduced agglutination of lymphocytes showed a similar sensitivity to cap inhibitors, although absolutely no capping of the PWM bound to the cells could be detected [4]. Indeed, as seen in table 2, the agglutination of lymph node and thymus lymphocytes could be readily induced by PWM at 50 ,ug/ml. Exptl Cell Res 82 (1973)

420 F. Loor

Table 2. P WM-induced lymphocyte agglutination Extent of cell agglutination Assay No agglutinin Inhibition Reversion

Lymphocyte source NLN NT NLN NT NLN NT

No inhibitor - or + +to++ + + + -t + +

to + -I- +

++++

14.5 %

NaN, 1O-2M

1O-4M CB

-

7% N.D.

-

27.5 % 74.5 %

-

19 96 - to + + + + +

34.5 % 78.5 %

3%

N.D.

63 %

i- +

53 %

-

(N.C.)

+ + + +

(N.C.)

+ + + +

6.5 % 13% 9%

(NC.)

PWM at 50 pg/ml. N.D., Not done. Other designations as for table 1.

That agglutination was totally inhibited by CB. Agglutinates of lymph node lymphocytes but not of thymus lymphocytes could be dissociated by CB into individual lymphocytes. NaN, had no marked effect both in the inhibition assay and in the reversion assay. When the microscope observation was made as described above for PHA, it was found that the PWM bound to the thymocytes was homogeneously distributed on the membrane (ring pattern) without any detectable spotting or capping [4]. There was absolutely no detectable difference whether

a cap inhibitor was present or not, whether the cells were in agglutinates or not (fig. 2). However, no binding of PWM could be detected on lymph node lymphocytes, although they could be agglutinated. Con A-induced agglutination

The agglutination of lymphocytes by Con A at 2.5 ,ug/ml was also inhibited or significantly reduced by NaN, and CB (table 3), despite the fact that, as judged by fluorescence microscopy, the initial amount of fluorescent ConA fixed by the cells was simi-

Table 3. Con A-induced lymphocyte agglutination Extent of cell agglutination Assay

Inhibition

Lymphocyte source NLN NS NT N TDL T TDL NLN NS

Reversion : ;DL T TDL

No inhibitor

1O-2M NaN,

1O-4M CB

- or + - or + f + or + or + +t-++ - or + - or + + + or ++ ++++

- or + (1) +

- or + (1) -

(1) (1) + + + (1) + (1) (1) (1) + + + (1)

ConA at 2.5 ,ug/ml. N S, normal spleen lymphocytes; N TDL, normal thoracic duct lymphocytes; T TDL, educated thoracic duct lymphocytes. (1) Depending on the mouse. Other designations as for table 1. Exptl Cell Res 82 (1973)

Lectin-induced lymphocyte agglutination

42 I

Table 4. Dose and temperature dependence of Con A-induced agglutination Sample Lymphocyte source

Extent of cell agglutination ConA dose Temperature (m/ml) (“C) 0

N LN

2.5 100

0 NT

2.5 100

0

37 0 37 0

37 0 37 0 37 0 31

No inhibitor

lo-” M NaN, .L (8.6) (13.4) (5.1) (17.9) (10.3) (98.7)

_

_ - or I .~.

+ + -k I-

- or -t- + or +-I -t -r 4 +

--

(5.9)

(6.5)

(3.7)

(2.3

!

(3.4) .~ (88.0)

(9.8)

$:l I

(8:8) (48.3)

or

(12.2) (7.0) (27.2)

(98.2)

L 2- ! !

(94.6)

(4.8)

(6.3) ---

lnhibition assay only. See table 1 for designations.

lar whether the cap inhibitors were present or not. Both thymocytes and lymphocytes from other sources bound similar amounts of Con A as judged by their equivalent cell membrane brightness. In absence of cap inhibitors and for low doses of ConA (l-10 pglml) the agglutinin could form caps on the lymphocyte membrane. There were striking differences in the ability of lymphocytes from various sources to be agglutinated by low doses of Con A. However, those differences disappeared when high ConA doses were used. For instance (table 4), when treated with ConA at 100 pg/ml, a dose known to ‘freeze’ the membrane [4, 10, 121, lymph node lymphocytes and thymocytes were equally well agglutinated. Such agglutination with high ConA doses, while still temperature-dependent, was not significantly NaN,-sensitive. In all the experiments described, the action of CB and NaN, in the inhibition assay was immediately reversible by one simple washing of the cells (which corresponded to only a lo-fold dilution of the inhibitor!); that removal of NaN, or CB allowed both

the fast agglutination of the cells and the capping of the bound Con A or PHA (no capping of PWM). DISCUSSION In contrast to the data recently reported by Nicolson [3] that would favour a completely passive agglutination theory (which might, in fact, be true when cells are treated with very large doses of agglutinin), we presented facts which rather support the existence of an active cell metabolism dependent step in the lymphocyte agglutination induced by low doses of &tins, whatever the nature of the actual physico-chemical interactions involved later in the maintenance of cell aggregation. From this and other studies [4], we can point out a few facts. Agglutination of lymphocytes can be obtained with doses of agglutinin low enough to leave the cells perfectly alive, and that are even mitogenic for some lymphocytes [I, 41. Thymocytes are more agglutinable and they pick up more PHA and much more PWM than do lymphocytes from other sources, Exptl Cell Res 82 (1973)

422

F. Loor

thus showing qualitative and/or quantitative differences in their glycocalix composition. However the binding of Con A is similar, both when estimated by fluorescence brightness and by more quantitative studies [13], and yet the thymocytes are much more agglutinable by ConA than spleen or lymph node lymphocytes. Thus no simple correlation can be made of agglutination to amount of lectin bound. Moreover, neither NaN, nor CB (cap inhibitors) interfered with the amount of membrane-bound agglutinins, whether lymphocytes were coated with agglutinin at 0°C or at 37°C. Then again the decreased agglutinability of the cells could not be simply due to decreasedamounts of agglutinin fixed on the cells. The agglutination of the lymphocytes by low agglutinin doses occurred in physiological conditions where the capping of agglutinin bound to the membrane was allowed and might actually occur. In non-capping conditions, agglutination was strikingly reduced. An important redistribution of the agglutinin might occur, however; no redistribution of PWM could be detected, but small spots of ConA and large patches of PHA were formed in the presence of NaN, or CB at room temperature or at 37°C. Although this would then fulfil the argument developed by Nicolson for the passive agglutination theory, i.e. agglutinin binding site clustering, still no agglutination occurred. Thus redistribution of lectin binding sites is not by itself sufficient to allow lymphocyte agglutination. The main argument against a simple passive agglutination theory as universal model of cell agglutination remains the observation that it was possible, at least with some cellagglutinin combinations, to dissociate agglutinates of lymphocytes into free lymphocytes by incubation with gentle shaking in the presenceof NaN, or more efficiently of CB. >l.Gell

Res 82 (1973)

Passiveagglutination may occur when cells are treated with high doses of lectin, since no sensitivity to NaN, was found for the agglutination of the lymphocytes with doses of 100 ,ug/ml ConA. As observed here and by others [14, 151 such agglutination was temperature-dependent, this being interpreted as a possible requirement for membrane fluidity to allow agglutinin binding sites clustering. However, when such high Con A doses are concerned, the membrane fluidity must be strongly reduced as membrane component redistribution is completely inhibited: no spotting of immunoglobulin receptors could be obtained [4, 10, 121and the ConA bound to the lymphocytes does not itself cluster into spots or patches [4]. If the agglutination of lymphocytes induced by low doses of lectins is not simply a passive agglutination due to direct cross-bridging of cells by agglutinin molecules, what can be the cell metabolism step(s) involved in an agglutination process that could be affected by NaN, and CB? Various possibilities can be considered: (I) Agglutination could require cell movement in order to get cell to cell contact; (2) agglutination could require capping of the agglutinin binding sites in order to uncover cryptic agglutinator sites; (3) agglutination could require microvilli formation by the cell in order to get their binding to other cells. In favour of the first suggestion is the fact that CB inhibits lymphocyte mobility probably by disrupting microfilament integrity and/or function [16]. NaN, may also affect cell movement as it is a metabolic poison with multiple sites of action. But, in fact, in our standard assay, cells were sedimented by centrifugation as a pellet, and kept at high concentration for observation. Thus, even in the absence of cell mobility there is a high probability of cell to cell contact. Moreover, one does not see why the CB and NaN,

Lectin-induced lymphocyte agglutination

sensitive metabolic step would still be required to keep the cell agglutinated as suggested by our reversion assay. The second suggestion would be supported by the fact that actually while inhibiting the agglutination, we did indeed inhibit the capping of the agglutinins and uncovering of cryptic agglutinator sites would then also be inhibited. The existence of such agglutinator sites was postulated by lnbar et al. [14]. However, PWM did not cap but agglutinated, and agglutination might precede PHA or Con A capping; and when Con A and PHA did cap, the caps were formed in contact with the other cells, leading to a much higher concentration of agglutinin in the zone of cell to cell contact than on the remainder of the cells. This does not fit with the cryptic agglutinator sites suggestion. The third alternative speculatesthat microvilli are involved in the agglutination and does not exclude the possibility that clustering of the agglutinin binding sites would very much increase the efficiency of the agglutination, as agglutinins have low affinity for their known ligand. Those clusters of agglutinin binding sites would be present on microvilli ends. This concept fits the best a series of facts obtained by ourselves and others. CB is known to inhibit the cell adhesion to glass and plastic surfaces and microvilli formation [17], presumably by damaging microfilaments (161. CB can also release lymphocytes that adhered to DNP-derived fibres (Kiefer, H & Loor, F. Unpublished), and can inhibit the T cell rosettes formed by human T lymphocytes with sheep red cells (Read, S & Loor, F. Unpublished), and it seemsreasonable to assume that microvilli are also involved in these processes. Moreover, drugs known to interact with microtubules, vinblastine and Colcemid, have been recently reported to inhibit the ConA induced agglutination of polymorphonuclear leucocytes

423

[18] and that of normal and transformed fibroblasts [19]. It is interesting to point out that vinblastine does not inhibit lymphocyte Ig receptors capping, even if used at doses 100 times higher than needed to inhibit Con A-induced agglutination (Loor, F. UnpuhIished), and this constitutes further evidence that the capping process itself is not involved in the agglutination process. Some microtubules as well as some microfilaments could then be involved either in the maintenance of a fixed planar distribution of the agglutinin binding sites or in the formation of protrusions on the cell surface. Both processes would require energy and it is quite feasible to postulate that NaN, could interfere with a triphosphatase activity of myosin-like protein involved in this microfilament/microtubule function [20]. The dissociation of clumps of lymphocytes by those drugs would be due to the dissociation of either microtubules or actin-like microfilaments that have either to keep a certain planar distribution of membrane components or to constitute some kind of backbone to the microvilli. This would lead either to randomisation of membrane components distribution or to microvilli fragility and breakage, and to loss of contact between the cells. Even in the hypothesis that an active step is involved in the agglutination process, the actual physico-chemical interactions involved in the maintenance of the cell aggregation are probably dependent on the cross-bridging of the cells by the agglutinin molecules. For that purpose, larger amounts of lectin bound to the membrane would then be more efficient. We feel that this would explain the lower sensitivity of thymus cells to the agglutination inhibitors. Indeed, when compared to lymphocytes from other sources and as judged by fluorescence microscopy. Exptl

Cell Res 82 (1973

424

F. Loor

thymocytes clearly bind similar amounts of ConA but about twice as much PHA and much more PWM than other lymphocytes. That higher agglutinability of thymus cells was also recently reported by Inbar et al. [15] and is probably related to a very different cell coat for thymus cells. Moreover, agglutinin binding sites clustering would enhance agglutinability of the cells. In fact the high agglutinability of the TTDL lymphocytes is likely to be due to the fact that 100% of them are mature T cells and according to Mandel [21], it seemsthat mature T cells would have an important membrane characteristic: the membrane intercalated particles detected by freeze cleaving procedures would not be evenly distributed as usual but strongly clustered into large patches [21]. As membrane particles are presumably connected with agglutinin binding sites [ 11, 221, clusters of membrane particles could form patches of agglutinin binding sites on the cell glycocalix (even if their size is too small to allow their detection as spots by fluorescence microscopy). It has been frequently reported that cells showing such a clustered distribution of agglutinin binding sites are much more agglutinable [3], and TTDL would represent an extreme case of that characteristic: they are readily agglutinated by doses of ConA that are 1 or 2 log lower than the doses of ConA required to agglutinate transformed cells or normal trypsinized cells [3] and that in fact would be highly toxic for the lymphocyte. But still the passive participation of the agglutinin binding sites, and of agglutinins as bridging agents, in this low-lectin dose induced agglutination is not evident. It should be pointed out that spontaneous clusters of cells always occur in control cell suspension in absence of any agglutinin, and that their formation is also strongly and reversibly inhibited by CB and NaN,. Another hypoExptl CeN Res 82 (1973)

thesis is that binding of agglutinin would be required to stimulate and allow formation of microvilli (or of other structures requiring microfilament activity and active metabolism) that would bear agglutinator sites that would stick to the membrane of other cells. If any kind of specific recognition were associated with that process, it would be similar to the well-known experiments of selective sponge cell reaggregation [23] or of segregation of chick and mouse embryo cells during reaggregation [24]. Thus I suggest that agglutination of cells by lectins could arise from two independent processes:active cell to cell recognition and passive cell to cell cross-linking by lectins. Depending on the composition of the cell glycocalix, on the lectin dose and on the presence or absence of agents likely to affect the cell metabolism, one or the other of the two proposed agglutination processes would be predominant: at low lectin doses there would be only active cell to cell recognition, while at high doses there would be only passive crosslinking of the cells by the lectins. The author is very much indebted to Dr J. Sprent who provided the normal and educated thoracic duct lvmuhocvtes. and to Dr G. Roelants for his helu during the preparation of the manuscript. The eicellent technical assistance of Miss Lena-Britt Haaa is very much appreciated. The author is charge 2; Recherches Honoraire from the Belgian National Funds for Scientific Research. Note added in proof Metabolic inhibitors of the capping process (dinitrophenol, NaN3 and CB) have been recently reported to inhibit tumor cell agglutination bv ConA and Ricinus communis aggluti& (Kaneko,-I, Satoh, H & Ukita. T. Biochem bioohvs res comm 48 (1973) 1504). Moreover it has been recently shown that CR inhibits and reverts nlatelet aggregation by blocking the formation of elongated p&dopodes needed for cell to cell adhesion (Boyle Kay. M M & Fudenberg. H H, Nature 244 (1973) 288). -

REFERENCES 1. Sharon, N & Lis, H, Science 177 (1972) 949. 2. Ginsburg, V & Kobaka, A, Structure and function of biological membranes (ed L J Rothfields) p. 439. Academic Press, New York-London (1971).

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Received March 13, 1973 Revised version received June 13. 1973

Exptl Cell Res 82 (1973)