The effect of lectins on the migration of lymphocytes in vivo

CELLULAR

IMMUNOLOGY

14, 66-79 (1974)

The Effect of Lectins MICHAEL

on the Migration

of Lymphocytes

in Vivol

SCHLESINGER AND EVELYNE ISRAEL

Department of Experimental Medicine and Cancer Research, The Hebrew Medical School, Jerusalem, Israel Received February

University-Hadassah

1.5,1974

The aim of the present study was to determine whether the cell-surface structures involved in the migration of lymphocytes to lymphoid organs are related to the structures on lymphocytes which interact with plant lectins. Cells from the spleen, lymph nodes, thymus, and bone marrow of A and C3H mice strains were labeled with SrCr, exposed in vitro to various concentrations of lectins, and injected intravenously to syngeneic recipients. Exposure of lymphocytes of various sources to Con A inhibited their migration to lymph nodes to a signtificantly greater extent than their migration to the spleen. Various concentrations of PHA inhibited the migration of lymphocytes to the spleen and lymph nodes to a similar degree. The migration of thyrnus cells was affected by lower concentrations of either Con A or PHA than the migration of the other cell types. Fucose-binding protein and wheat germ agglutinin failed to affect lymphocyte migration. The inhibitory effect of Con A could be abrogated by a-methylmannopyranoside added either simultaneously with Con A or following exposure of the cells to Con A. In contrast, the inhibitory effect of PHA could not be abolished by the addition of N-acetyl-galactosamine. Secondary transfer experiments with lymphocytes localizing in the spleen and lymph nodes of primary hosts indicate that Con A does not eliminate a subpopulation of lymph-node-seeking lymphocytes, but rather interferes specifically with the process involved in the localization of lymphocytes to lymph nodes.

INTRODUCTION Recirculating lymphocytes are endowed with a distinct capacity to seek out the lymph nodes and to localize there. It is not clear what factors are involved in the migration of lymphocytes to lymph nodes, but this process seems to depend on the presence of distinct glycoproteins on the membrane of the lymphocytes. Gessner and his colleagues showed that lymphocytes lose their capacity for localization in lymph nodes upon exposure of the cells to glycosidase (l), trypsin (2), or neuraminidase (3). Zata et al. (4) demonstrated recently that treatment of lymphocytes with potassium periodate, under conditions leading to the oxidation of cellsurface carbohydrates, inhibited the migration of lymphocytes to lymph nodes. Attempts have been made to correlate the cell-surface structures involved in the migration of lymphocytes with known cell-surface antigens (5, 6). Thymus cells localizing in lymph nodes were found to possess a high H-2 antigenicity, and to be 1 Supported by USPHS Research Agreement No. 06-048-I. A preliminary report was presented at the Joint Meeting of European Societies for Immunology, Strasbourg, France, September 4-7, 1973. 66 Copyright All rights

Q 1974 by Academic Press, Inc. of reproduction in any form reserved.

EFFECT

OF LECTINS

ON LYMPHOCYTE

MIGRATION

67

resistant to the cytotoxic activity of guinea pig serum (GPS). Spleen-seeking thymus cells had the opposite antigenic properties (6). Both H-2 antigens (7, 8) and the antigen reactive with GPS (9, 10) are known to contain a considerable amount of carbohydrate components. In the present study yet another approach was used for the characterization of the cell-surface glycoproteins involved in the migration of lymphocytes. Plant lectins, such as concanavalin A (Con A) and phytohemagglutinin (PHA) , are 51Chromium-labeled thymus, known to react with specific carbohydrates (11). spleen, or lymph node cells were exposed in vitro to various plant lectins, and their pattern of migration in Z&JOwas determined upon injection into syngeneic hosts. MATERIALS

AND

hTETHODS

Mice Mice of the A/J and C3H/Crgl inbred strains were used both as donors of lymphoid cells and as syngeneic recipients. Lectins Concanavalin A (Con A) was purchased a stock solution containing 38 mg Con A/ml Just prior to use, the Con A stock solution water, so as to yield an isotonic solution. Freeze-dried phytohemagglutinin (PHA) Research Laboratories, Beckenham, England, 5.0 ml sterile distilled water.

from Miles Yeda, Rehovot, Israel, as in saturated sodium chloride solution. was diluted 1 : 38 in sterile distilled (MR-10) , purchased from Welcome was reconstituted by the addition of

Fucose Binding Protein Fucose binding protein (FBP) was purchased from nliles Yeda, Rehovot, Israel. The stock solution contained 37 mg FBP/ml. Whmt Germ Agglutik Wheat germ agglutinin (WGA) was obtained from the late Dr. Michael Kapeller and from Professor F. Doljanski (12). Sugars a-Methyl-u-mannopyranoside (a-MM), ;V-acetyl-n-galactosamine, and N-acetyln-glucosamine were obtained from Pfanstiehl Laboratories, Waukegan, TT,. Radioactive Labeling of Lynphoid

Cells

Suspensions of thymus, spleen, and lymph nodes were prepared as described previously ( 13). Lymphoid cells were labeled with 51Cr by incubation with isotonic sodium chromate solution (Radio-chemical Center, Amersham, Bucks.) according to the method of Wigzell ( 14). Details of the technique were described previously ( 15). Ked blood cells present in the spleen and bone-marrow cell suspensions were lysed by exposure to 0.83% NH4Cl according to the method of Boyle (16), prior to labeling with 51Cr.

68

SCHLESINGER

AND

ISRAEL

Exposure of Labeled Cells to Lectins 51Chromium-labeled lymphocytes were washed four times in normal saline to remove any free label. The cells were suspended in normal saline at a concentration of (40-80) X lo6 cells/ml. Volumes of 0.5 ml of cell suspension were delivered into plastic test tubes (Packard), and dilutions of either Con A or various lectins were added. In experiments with Con A, 0.05 ml of serial fourfould dilutions of Con A in normal saline was added to each test tube. The concentrations of Con A given in the results refer to the amount of lectin added to 0.5 ml of the labeled cell suspension. Tenfold serial dilutions of PHA in normal saline were set up, and 0.1 ml of the PHA dilutions was added to each test tube. The dilutions of PHA given in the results refer to the final dilution of the lectin in each test tube. Control cell suspensions contained saline alone. Following the addition of the lectins, the suspensions were incubated at room temperature for 30 min. Just prior to injection of the labeled cells, 0.05 ml fresh, normal, syngeneic mouse serum and 1.0 ml saline were added to each test tube. A volume of 06-0.7 ml of the suspensions was injected intravenously into syngeneic mice. Competition Experiments 51Chromium-labeled thymus cells were suspended in 0.5 ml normal saline. To the cell suspension was added 0.12 pg Con A and 0.5 ml of a 0.15 M solution of a-methyl-n-mannopyranoside (a-MM). The mixture was left for 30 min at room temperature prior to its injection into syngeneic recipients. Controls received suspensions containing either Con A or (y-MM alone. In competition experiments with PHA, %r-labeled thymus cells were suspended in 0.5 ml saline, to which was added 10 pl PHA and 0.5 ml of a 0.1 n/r solution of N-acetylgalactosamine in saline, or 0.5 ml of 0.1 M solution of N-acetylglucosamine in saline. The mixture was incubated for 30 min at room temperature and was then injected to syngeneic recipients. Control suspensions contained either PHA alone, or the acetyl-amino derivatives of one of the sugars. E&ion

Experiments

Elution of Con A was carried out according to the procedure of Anderson et al. (17). Volumes of 0.5 ml of Yr-labeled thymus cells were incubated with 0.5 pg Con A for 30 min at room temperature. The suspensions were washed once in saline, and resuspended in 1.0 ml of a 0.1 M solution of (U-MM in saline. The mixture was incubated for 1 hr at 37°C and was then washed twice in 0.1 M a-MM solution. The cells were finally suspended in normal saline and injected into syngeneic recipients. Controls received suspensions incubated either in Con A alone or in a-MM. The Distribution

of Labeled Cells

Twenty hours after the intravenous injection of labeled lymphoid cells, the recipients were killed, and their lymph nodes (axillary, brachial, inguinal, and mesenteric), spleens, and livers were dissected out. The radioactivity of each organ was determined in a well-type scintillation counter (Auto-gamma, Packard). The radioactivity detected in each organ was calculated as the total radioactivity injected.

EFFECT

OF LECTINS

ON LYMPHOCYTE

60

MIGRATION

1

TABLE

THE MIGRATION OF ~'CR-LABELED LYMPHOID CW.W SIISPENDED IN %ylS hlice injected Strain

Source of cells injected

Lyrrlph nodes

SO.

i\ A A

23 21 16 .z

~--_____ Lymph nodes Spleen Thymus Bone marrow

C3H C3H C3H C3H

12 1.5 33 4

Lymph nodes Spleen Thymus Bone marrow

A

a Percentage

Ixxalization __ ._ -_~_~-

______

of the total radioactivity

--___-

-

in .~. - --.-I_

SpltTn

~.. Liver

~~

11.47 3.78 1.89 0.39

zk xt f f

0.62” 0.16 0.08 0.04

15.15 13.41 12.09 6.74

* f * i

0.49 0.74 0.34 1.13

19.85 31.42 27.31 29.09

f f f r!z

0.51 0.97 0.6’) 0.21

14.44 3.17 1.99 0.38

f * I-t f

1.07 0.23 0.12 0.05

14.86 12.41 12.48 9.59

zt f h f.

1.08 0.52 0.62 0.46

17.49 32.07 23.47 26.88

f h f f

0.45 0.91. 0.78 2.15

injected

(mean f

SE)

Such data are presented in Table 1 and in Tables 9-12. In animals receiving cells incubated with lectins, the residual radioactivity detected in each organ is expressed as a percentage of the relative radioactivity found in the same organ in mice receiving control cells, not exposed to lectins, according to the formula ($% radioactivity of total radioactivity injected, _____ lectin-treated cells) (% radioactivity of total radioactivity injected, control cells)

Statistical

x loon.

Ana.lysis

The significance of differences among various mean values was evaluated with the help of Student’s t test. RESULTS The Migration

of Lpzphocytes

Incubated

in Norwzal Saline

Table 1 shows the pattern of migration of Yr-labeled lymphocytes which had been kept in normal saline for 30 min at room temperature without the addition of any lectin. As in previous studies (5, 18, 19) the proportion of lymph-node-seeking lymphocytes was highest by far in suspensions of lymph node cells. Spleen cell SWpensions contained a higher proportion of lymph-node-seeking cells than thymus cell suspensions, whereas bone marrow contained the lowest proportion of such cells. The proportion of cells localizing in the spleen was similar in suspensions of lymph nodes, thymus, and spleen but was relatively lower in suspensions of bone marrow cells. The EfSect of Con A on the Migration Thpms

migration migration

of Lymphocytes

cells. The exposure of thymus cells to Con A markedly inhibited their to the spleen and lymph nodes. In strain A mice, Con A inhibited the of thymus cells to the lymph nodes to a significantly greater extent than

70

SCHLESINGER

AND

TABLE

ISRAEL

2

THE EFFECT OF CON A ON THE LOCALIZATION OF WIGLABELED IN ORGANS OF SYNGENEIC RECIPIENTS Mice injected Strain

No.

Concentration of Con A (rcdO.5 ml)

Localization Lymph

nodes

THYMUS

in Liver

Spleen

A

6 6 6 6

2 4 AT ih

4.35 8.92 16.43 47.16

f f f f

1.2P 1.94 2.00 4.27

15.01 23.46 45.62 83.21

* 1.80 f 4.06 f 4.38 f 6.47

85.38 101.46 99.81 91.92

f f St f

3.19 8.04 3.46 3.43

C3H

4 4 6 6

2 i & 2z

10.00 20.09 42.78 73.67

f i f f

4.31 2.26 7.56 8.03

12.98 30.56 55.20 80.48

f f f f

92.49 105.66 100.38 96.55

f f f zk

3.71 8.74 16.26 10.28

a The localization of Con A-treated cells is expressed control cells not exposed to Con A (mean i SE).

3.35 9.37 3.99 6.22

as a percentage

of the localization

of

the migration to the spleen (P < 0.005) (Table 2). This was evident at all Con A dilutions studied. As low a dose as 0.016 pg of Con A (per 40 x lo6 cells) still caused a striking inhibition of the migration of strain A thymus cells to the lymph nodes. In contrast to its effect on strain A thymus cells, Con A inhibited the migration of C3H thymus cells to lymph nodes and spleen to a similar extent (Table 2). The proportion of thymus cells localizing in the liver was slightly reduced following exposure to 2 ,pg of Con A, but lower doses had no effect on the localization of cells in the liver. Lymph node cells. Concanavalin A affected the migration of lymph node cells only at the two highest concentrations tested (2 and 0.5 ,pg). The localization of lymph node cells in lymph nodes was strongly inhibited by these concentrations of TABLE

3

THE EFFECT OF CON A ON THE LOCALIZATION OF 61Cr-LABELED LYMPH NODE CELLS IN ORGANS OF SYNGENEIC RECIPIENTS Mice injected Strain

A

C3H

No.

Concentration of Con A (do.5 ml)

Localization Lymph

nodes

in Liver

Spleen

10 9 8 2

2 3 5% &

12.46 f 31.96 f 72.93 f 86.49

2.50” 4.87 5.06

40.42 f 75.47 f 97.73 f 106.57

8.77 5.62 6.37

147.74 f 121.75 f 104.34 f 89.40

8.68 4.33 3.28

4 4 4

2 Q AT

11.67 f 43.09 f 72.46 f

1.21 3.24 9.24

42.49 f 92.14 f 98.82 f

9.28 9.02 8.65

175.23 f 125.85 f 104.61 f

9.83 8.11 6.34

6 The localization of Con A-treated cells is expressed as a percentage cells not exposed to Con A (mean f SE).

of the localization

of control

EFFECTOFLECTINSON

LYMPHOCYTE TABLE

71

MIGRATIOS

4

THE EFFECT OF CON A ON THE LOCALIZATION OF 61C~-L~~E~~11 SPLEEN CELLS INORGANS OF SYNGENEIC RECIPIENTS

Strain

A

C3H

Localization

Concentration of Con A (pg:O.S ml)

Mice injected NO.

10 8 7

2 f 72

2 3

2 t

Lymph

SplWll

1,iver

38.96 zk 8.94 62.33 i 8.25 94.64 zt 4.86

110.44 f 12.33 i.50 109.74 i 104.20 z!z 5.20

48.58 f 99.35 f

4.06 112.40 f 118.16 + 12.58

nodes

15.66 f 3.85s 30.92 f 9.62 77.15 & 6.86 14.13 i 49.82 f

0.62 1.75

in

0.99 3.43

(’ The localization of Con A-treated cells is expressed as a pcrccntage cells not exposed to Con A (mean s SE).

of the localization

of coutr~ll

Con A, whereas the localization in the spleen was affected to a significantly lesser extent (P < 0.01) (Table 3). This was true for mice of both the A and C3H strains. Unlike the findings obtained with thymus cells, those concentrations of Con A which affected the migration of cells elicited a marked increase of radioactivity detected in the liver (Table 3). Spleen cells. The effect of Con A on spleen cells paralleled its effect on lymph node cells. Here too, the migration stream to lymph nodes was inhibited to a significantly greater extent than the spleen-seeking migration stream (P < 0.001) (Table 4). Comparable results were obtained with spleen cells of both strains of mice tested. Exposure to Con A slightly increased the amount of radioactivity localizing in the liver. Bone marrow cells. The effect of Con A on the localization of bone marrow cells was studied only in a small number of mice. Concanavalin A tended to inhibit the

TABLE

5

THE EFFECT OF CON ,I ox 'I‘HE LOCALIZATION OF %A,ABELED CELLS ~ORGANSOF SYNGENEIC RECIPIENTS Mice injected Strain

NO.

Concentration of Con A (do.5 ml)

Localization Lymph __---

-

nodes Spleen ~~___ __.__

BONE MAHIUN

in Liver ~_

.il

2 2 2

2 + 116

23.46” 53.65 96.34

31.40 80.51 107.85

101.67 135.07 123.60

C3H

2 2

2 f

42.68 57.32

41.93 75.05

6X.45 118.18

a ‘The localization of Con A-treated cells is expressedas a percentageof the IocaIization of c~rtro~ cells not exposed to Con A.

72

SCHLESINGER

AND ISRAEL

TABLE

6

THE EFFECT OF PHA ON THE LOCALIZATION OF %R-LABELED IN ORGANS OF SYNGENEIC RECIPIENTS Mice injected Strain

PHA dilution

Localization

No.

Lymph

nodes

in

Spleen

A

4 8 4 2

l/50 1 /so0 1 /so00 l/25,000

11.29 & 2.79a 28.93 f 2.97 71.97 f 5.46 93.95

11.73 i 29.29 f 59.05 f 99.48

C3H

2 2 2

l/500 1/so00 l/25,000

38.87 58.33 63.88

36.35 74.16 74.91

a The localization of PHA-treated cells not exposed to PHA (mean f

THYMUS CELLS

Liver

2.97 6.40 4.94

88.26 f 110.18 f 116.57 f 116.24 83.23 61.15 67.66

cells is expressed as a percentage SE).

of the localization

cells in the lymph nodes to a slightly of bone marrow than their localization in the spleen (Table 5).

localization

The Efect of PHA on the Migration

6.72 4.08 11.88

of control

greater extent

of Lymphocytes

Thymus cells. Exposure of thymus cells to 1: 50 or 1: 500 dilutions of the standard PHA solution markedly inhibited their migration to either the lymph nodes or the spleen (Table 6). No significant difference was observed between the effect of PHA on both migration streams. For each concentration of PHA the inhibitory effect was more pronounced with thymocytes of mice of the A strain than with thymocytes of the C3H strain. The localization of label in the liver of C3H mice was reduced by all concentrations of PHA studied. In contrast, in strain A mice only the highest concentration of PHA tested reduced the amount of label recovered in the liver, while all other concentrations of PHA elicited a slight increase. Lymph node cells. Exposure of lymph node cells to PHA inhibited their migration to either the lymph nodes or the spleen to a similar extent (Table 7). The TABLE

7

THE EFFECT OF PHA ON THE LOCALIZATION OF 51C~-L~~~~~~ CELLS IN ORGANS OF SYNGENEIC RECIPIENTS Mice injected Strain

Localization

PHA dilution Lymph

No.

nodes

A

4 4 4

l/SO l/SO0 1 /so00

28.21 f 1.84” 54.51 * 5.25 98.64 f 8.50

C3H

2 2 2

l/50 l/500 1 /so00

20.68 69.95 94.85

6 The localization of PHA-treated cells not exposed to PHA (mean f

in Liver

Spleen 36.24 f 65.75 f 101.55 f 31.48 73.95 99.32

cells is expressed as a percentage SE).

LYMPH NODE

5.99 4.37 6.08

92.61 f 141.97 f 112.53 f

10.68 8.83 11.31

117.76 182.35 118.21 of the localization

of control

EFFECTOFLECTIiXSON

LYMPHOCYTE

7.3

MIGRATION

sensitivity of lymph node cells to the inhibitory effect of PHA was slightly less than that of thymus cells. A 1: 500 dilution of PI-IA, which had a marked inhibitory effect on the migration of thymus cells, had only a weak effect on the migration of lymph node cells. Phytohemagglutinin had a variable effect on the localization of label in the liver, but usually the amount of label detected in the liver of mice injected with PHA-treated lymph node cells was higher than that detected after the administration of untreated cells. Spleen rells. The inhibitory effect of PHA on the migration of spleen cells RX very similar to its inhibitory effect on the migration of lymph node cells (Table= 8). Here too, PI-IA affected the lymph-node-seeking and spleen-seeking subpopulnt ions of spleen cells to a similar degree. The Efiert

of FBP

and WGA

on the Migration

of A/J

LgmFhoid

Cells

Strain A thymus and lymph node cells were exposed in vitro to 1: 8 and 1: 16 dilutions of a preparation of wheat germ agglutinin (WGA) . No significant effect of WGA on the migration of cells to either the lymph nodes or the spleen could be observed. Exposure of strain A thymus cells to 50-100 pg fucose-binding protein (FBP) had only a slight and nonsignificant inhibitory effect on the migration of the cells to the lymph nodes, but failed to affect the migration of cells to the spleen. nnd elution e.rpeerimmts

Competition

The inhibitory effect of Con A on the migration of lymphocytes i?z viva could possibly result from a toxic effect of Con A on the cells during their in vitro exposure to Con A. This possibility seemed unlikely in view of the fact that the low concentrations of Con A used had no visible cytotosic effect on the cells. as determined by the trypan blue exclusion test. It could be argued, however, that the in vitro exposure to Con A had a cytotoxic effect on the distinct subpopulation of lymphocytes which migrates to the lymph nodes. To study this problem, thymus cells were incubated with either Con A alone (1 : S $/0.5 ml) or with a-n/N (I 5 mg/0.5 ml) or with both (Table 9). The addition of CU-MM to Con A almost con+ pletely prevented the inhibitory effect of Con A on cell migration. In another series of experiments, Con A was eluted from the surface of thymus

Llic-e injected ~- -St rain ~~

PH.\ dilution

No.

I~orali~ation Lymph

nodes

in

Spleen

laivrr

~~~~. ___~_ A

C3H

4

l/SO

7 4

l/500 1 ,/so00

2 2 2

l/SO l/500 l/5000

u The localization of PHA-treated cells not exposed to PHA (mean f

28.29 & 1.W 72.90 f 5.09 97.15 f 12.70 36.05 106.32 119.13

47.22 f 84.37 f 97.17 f 34.X 84.02 111.81

cells is expressed as a percentage SE).

4.88 6.61 8.28

89.33 i 4.12 94.57 * 1.88 92.92 z!z .Z.% 107.23 106.82 95.92

of the localization

of control

74

SCHLESINGER

AND

TABLE

ISRAiiL

9

THE COMPETITIVE EFFECTOFWD-METHYLMANNOPYRANOSIDEAND CONCANAVALIN A ONTIIE MIGRATION OF A/J THYMUS CELLS Incubation

with

Localization Lymph

Saline Con A (0.125 pg) or-MM (15 mg) Con A + (r-MM a Percentage

of the total radioactivity

nodes

1.75a 0.55 1.56 1.39 injected

in

Spleen

Liver

9.35 7.79 13.19 10.90

36.98 33.74 30.49 28.49

(mean of results obtained

in two mice).

cells by the addition of a-MM after the cells had already been incubated with Con A alone for 30 min. Cells which had been exposed to Con A were found to regain their capacity for migration to lymph nodes and spleen following incubation with a-MM for 1 hr at 37°C (Table 10). It is clear, therefore, that Con A does not prevent the migration of cells by killing them in z&o. Attempts were made to abrogate the inhibitory activity of PHA on cell migration by a competitive effect of N-acetyl-galactosamine or N-acetyl-glucosamine. Thymus cells exposed to PHA (diluted 1: 50) in the presence of 0.15M of either of these sugars had the same diminished capacity of localizing in the spleen and in the lymph nodes, as cells exposed to PHA alone (Table 11). The Effect of Con A on Serially Transferred Cells The competition and elution experiments showed that Con A did not kill lymphnode-seeking cells in vitro. It was possible, however, that prolonged contact with Con A may specifically eliminate in viva the population of lymph-node-seeking cells. Alternatively, Con A could block carbohydrate receptors on the cell membranes, TABLE

10

THE EFFECT OFT-D-METHYLMANNOPYRANOSIDE ONTHE MIGKATION OF CON A-COATED THYMUS CELLS Strain

Incubation

Localization

mixture

Lymph

in

Spleen

Liver

nodes A A A A

Saline Con A (0.5 pg) a-MM (15 mg) Con A-coated cells + ru-MM

1.62a 0.45 2.05 2.04

10.63 2.14 9.86 8.89

27.85 20.24 30.51 30.47

C3H C3H C3H C3H

Saline Con A (0.5 pg) a-MM (15 mg) Con A-coated cells + or-MM

1.41 0.49 1.44 1.57

19.20 7.47 14.09 20.03

22.70 28.02 20.90 21.05

0 Percentage

of the total radioactivity

injected

(mean of results obtained

in two mice)

EFFECT

OF LECTIIGS

Or; LYMPHOCYTE

TABLE

7’ I .’

MIGRATIOS

11

‘I‘tw EFFIK~ OF N-ACETYL-AMINO SC’GAKS ANI) I’BBWHEMAM ON THE MIGRATION OF C3H THYMUS CELLS incubation

.I.UUNIN

mixture Lymph nodes

Saline PHAb N-acetyl-galactose N-acetyl-glucose PHA + N-acetyl-galactose PHA + N-acetyl-glucose

1.34J 0.49 1.22 1.12 0.46 0.32

11.85 2.94 12.40 9.75 2.91 2.23

19.77 21.53 24.42 26.05 27.61 23.92

” Percentage of the total radioactivity injected (mean of results obtained in two mice). b PHA dilution, l/SO: N-acetyl-galactose, 0.15 M; N-acetyl-glucose, 0.15 ;2f.

which are directly involved in the migration of cells into lymph nodes. To distinguish between these two possibilities, the effect of Con A was studied on lymphocytes which localized in the lymph nodes of primary hosts, and which are known to constitute a relatively homogeneous population of recirculating cells ( 181. ~lCl~romium-labeled lymph node or spleen cells were injected into primary hosts ; 20 hours later the primary hosts were killed and suspensions were preparetl from their lymph nodes. Part of the suspension was exposed to Con A (0.5 &20 x lo6 cells), prior to injection into secondary hosts, while another part of the suspension was injected without further treatment. The localization of untreated lymph node cells in the lymph nodes of secondary hosts was significantly higher than their localization in the lymph nodes of primary hosts (Table 12). When the cells localizing in the lymph nodes of primar! hosts were exposed to Con A, their migration to the lymph nodes of secondary hosts was significantly inhibited, whereas their migration to the spleen of these hosts was unaffected.

TABLE

12

THE EFFECT OF CON A ON THE LOCALIZATIOP; OF WR-~.AFSRLI.X) LYMPHOCYTES IN SECONDAKV SYNGI?NEIC HOSTS Original

inoculum con A ;,dded --

Cells

Lymph node cells Spleen cells Spleen cells Spleen cells ” Percentage

+

of the total

Source of cells in primary host

Localization in primathost - ---~

Lymph nodes

Spleen

12.65’ 2.89 3.35 0.88

15.89 12.51 16.26 10.01

radioactivity

Lymph Spleen Lymph Spleen

iniected

(mean

nodes nodes

Localization in secondary hosr ~. ~~- ~ ~~----~~ ~~-\Vithout Con X Con ~4 ,idded Lymph nodes

Spleen

I<>mph nodes

20.28 2.81 12.78 2.35

18.99 11.29 16.80 9.16

7.85 0.80 3.97

of results obtained

in 2-4 mice).

Spkcn

21.82 10.0.3 13.08

76

SCHLESINGER

AND

ISRAEL

Spleen cells which localized in the lymph nodes of primary hosts showed a fourfold increase in their capacity of localizing in the lymph nodes of secondary hosts (Table 12). Here again, exposure of lymph-node-seeking cells to Con A had a significant inhibitory effect on their localization in lymph nodes of secondary hosts, but did not affect their localization in the spleen. Spleen cells which localized in the spleen of primary hosts had precisely the same capacity of localizing in the lymph nodes of secondary hosts as the initial spleen inoculum. Exposure of spleen cells localizing in the spleen of primary hosts to Con A affected only their localization in the lymph nodes of secondary hosts, but did not affect their splenic localization. In further experiments, 51Cr-labeled spleen cells were exposed to 0.5 lug Con A/25 X lo6 cells prior to injection into primary hosts. Twenty hours later, suspensions were prepared from the spleens of the primary hosts, and injected without further treatment into secondary hosts. As seen in Table 12 these cells had a normal capacity of migrating into either lymph nodes or the spleen of the secondary hosts. DISCUSSION In the present study the in vitro exposure of lymphocytes to either Con A or PHA was found to inhibit their capacity of localizing in the lymphoid organs of syngeneic recipients. Phytohemagglutinin inhibited to a similar extent the migration of lymphocytes to either the lymph nodes or the spleen, Concanavalin A had a significantly stronger inhibitory effect on the lymph-node-seeking as compared to the spleen-seeking migration stream. During the preparation of this manuscript similar observations on the effect of Con A were reported (20). There are many, not mutually exclusive, ways by which lectins could affect the migration of lymphocytes : (1) Cells exposed to lectins could be rapidly eliminated from the circulation through phagocytosis by the reticuloendothelial system. (2) Exposure of lymphocytes to lectins may lead to their killing either in vitro or in z&vu. (3) Upon exposure to lectins, lymphocytes may undergo blastic transformation, which in turn may impair their capacity for migration. (4) The attachment of lectins to the cell membrane may alter its biophysical properties, and may affect the cell-surface charge and the fluidity of the membrane. (5) Lectins may interact with specific sites on the membrane which are involved in migration of lymphocytes. Jacobson and Blomgren (21) have shown that activated T lymphocytes, tested 5 days after exposure to allogeneic antigens, show a diminished capacity for localizing in lymph nodes. They suggested that following blastoid transformation T cells may stop synthesizing the specific cell-membrane receptors necessary for localization in lymph nodes. It is not likely that blast cell transformation is involved in the effect of Con A or PHA on lymphocyte localization in the present study, since cells triggered by either of these lectins should show a similar pattern of defective migration. Moreover, the inhibitory effect of lectins on lymphocyte migration observed in the present study occurred within 20 hr, before any considerable blast transformation would have taken place. The present study does not allow an unequivocal assessment of the mechanisms involved in the effect of PHA on lymphocyte migration. The effect of PHA prob-

EFFECT

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57

ably is not due to its blastogenic effect, for the reasons outlined above. Moreover, PHA inhibited the localization of lymphocytes in the spleen and in the lymph nodes to the same extent, yet lymph-node-secl;ingle-secl~ing cells wcrc found to respond more vigorously to PHA than spleen-seeking cells (22, 2.3) It is not clear to nhat estent PHA affects lymphocyte migration through its interaction with carbohydrates on the membrane of lymphocytes. The addition of Wacetyl-galactosamine failed to abrogate the inhibitory effect of PHA on cell migration. Previous studies ll:rvr sh~vn that, whereas this sugar inhibits the agglutination of the lymphocvtrs 10 PHA (24)) it does not inhibit the blastogenic effect of PJ-IIJ (23 ) . What is the mechanism of action of Con A on the migration of lymphocytes T The fact that a-methyl-mannopyranoside (a-MM) counteracts the inhibitory effect of Con A clearly shows that Con A does not kill the affectecl lymphocytes during their in vitro esposure to the lectin. It also indicates that Con A affects the migration of lymphocytes by attaching to carbohydrate-containing moieties on the cell surface. The normal migration capacity of the Con A-treated lympbocytt~s \zllirh had lodged in the spleen of primary hosts indicates that the splenic localization ot’ Con A-treated cells does not represent phagocytosis of Con h-coated cells b!. the spleen. The different localization of Con A-treated cells in the spleen and in the lymph nodes, therefore, does not reflect different phagocytic elimination of thr cells by these organs. The possibility that Con A eliminates a distinct subpopulation of ~ylllphCJc~-tcS in 7iz'o is ruled out by observations on the migration of lymphocytes in secondar! hosts. T.ymph node cells which migrate to the lymph nodes of primary syngeneic hosts constitute a relatively homogeneous population of recirculating cells (~1X). If Con A acted by eliminating the lymph-node-seeking cells. one would expect that, when cells M.hich had localized in the lymph nodes of primarv hosts are exposed to Con A, their migration to either the spleen or the lymph nodes of sccontlarl hosts would be inhibited to the same extent. The results showed. however, that C‘o11;\ affected only the migration of cells to the lymph notles of secondar>- hosts, hut failed to affect their migration to the spleen of secondary llosts. Further evi(lcnc-e that Con A does not eliminate in vitro the lymph-node-seeking population QXS obtainrd in studies on the migration of Con A-treated splenic lymphocytes which ll;ld localized in the spleen of primary hosts. These lymphoc~~trs showed a l)eriectlq normal capacity of migrating not only to the spleen hut also to the lymph 110t1es of secondary hosts. Since all these experiments indicate that Con A doex not eliminate the population of lymph-node-seeking cells, it seems reasonable to ~~11elude that Con A has a preferential inhibitory effect on the migration of lymphocytes to the lymph nodes, at concentrations which do not affect migration to the spleell. The inhibitory effect of Con A on the migration oi lymphucytcs to the Iympll nodes may be a nonspecific consequence of the attachment of the lectin to the cell membrane. At higher concentrations than those usctl in the present stucly, (‘on 4 impairs the free fluidity of the cell membrane and inhibits “capping” of cell-surface determinants (26, 27). Alternatively, it may be suggeste(1 that Con I\ hinds specifically to carbohydrate-containing receptors ~vllicll determine the “botni~~~” of lymphocytes to lymph nodes. Several studies suggest the importance of carbohydrate-containing cell surface components for the distinctive localization of lymphocytes in lymph nodes. 17xposure of lymphocytes to trypsin transiently inhibits their migration to the lymph

78

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nodes without significantly changing their localization in the spleen (2). Trypsinization seems to affect predominantly the cell-surface glycoproteins (28). Treatment of lymphocytes with potassium periodate inhibits to a greater extent their migration to lymph nodes than to the spleen (4). Potassium periodate has been shown to oxidize cell-membrane glycoproteins containing sialic acid (29). Lymphocytes may bind between 3 X 106 and 10 x 10s molecules of Con A per cell (20-32), but maximal mitotic stimulation of lymphocytes can be obtained when Con A binds to only 3% of the Con A-binding sites on the cell membrane (30). In the present study relatively low concentrations of Con A were found to affect lymphocyte migration. It can be calculated that lo6 molecules of Con A per cell sufficed for inhibition of the migration of lymph node cells, whereas the migration of thymus cells was inhibited by as few as 3 x lo3 Con A molecules per cell. These concentrations of Con A are far lower than those required for “capping” of Con A-binding sites (33). There seems to be some parallelism between the capacity of lectins to inhibit the migration of lymphocytes and their ability to induce blast transformation at suitable concentrations. Phytohemagglutinin and Con A have a strong blastogenic effect on lymphocytes and are highly effective inhibitors of the migration of cells. In contrast, WGA and FBP, which in the present study had no effect on the migration of lymphocytes, were previously found to lack mitogenic effect on lymphocytes (34). Trypsinization of lymphocytes inhibits their migration to lymph nodes (2) and likewise abolishes their capacity of responding to PHA (23). Exposure of lymphocytes to potassium periodate inhibits the migration of cells (4) but also elicits blast transformation of the cells (35). It seems possible, therefore, that closely related carbohydrate-containing cell-surface structures, if not identical constituents, are involved in the response of lymphocytes to mitogens and in their distinctive localization in the lymphoid system. REFERENCES 1. 2. 3. 4.

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MIGRATIOS

7’)

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