The nature of cellular agglutinins of Androctonus australis (Saharan Scorpion) serum

DEVELO~NTAL AND COMPARATIVE I ~ O L O G Y , Vol. 3~ pp. 429-440, 1979. 0145-305X/79/030429~11502.00 Printed in the USA. Copyright (c) 1979 Pergamon Press Ltd. All rights reserved.

THE NATURE OF CELLULAR AGGLUTININS OF ANDROCTONUS AUSTRALIS (SAHARAN SCORPION) SERUM

Elias Cohen I, George H.U. Ilodi I, Z. Brahmi 2 and J. Minowada 3, Department of Laboratory Medicine I, 3 Department of Immunology and Immunochemistry , Rmswell Park Memorial Institute, Buffalo, NY, 14263, ~.S.A. 1'3 Institute Pasteur D'Algerie, Algiers, Algeria

ABSTRACT

The nature of cellular agglutinins of Androctonus australis (Saharan Scorpion) serum with selected comparisons with agglutinins of Limulus ~olyphemus (Horse-shoe crab) has been studied. Human peripheral chronic lymphocytic leukemic lymphocytes were agglutinated by Androctonus to higher titers and scores than peripheral normal lymphocytes. Androctonus agglutinins were able to differentiate more significantly between human peripheral lymphocytes than Limulus agglutinins. Human erythrocytes were agglutinated by Androctonus serum with no significant ABO differences. Hemagglutination inhibition of human erythrocytes by whole Androctonus and Limulus sera was demonstrated by selected sugars suggesting that Androctonus agglutinin receptors have different affinity and a different specificity for sugars than do Limulus agglutinins. Androctonus and Limulus sera may be useful, therefore, in identifying leukemic or "neoplastic" human cells, as well as studying topographic changes in normal and leukemic lymphocyte cell membranes. INTRODUCTION

Hemagglutinins in the serum of Limulus polyphemus, a marine arachnoid called the "Horse-shoe crab," were first described by Noguchi (13) as early as 1903. Later, Cohen et al (3-8) described experiments on Limulus agglutinins with human erythrocytes and lymphocytes; Cohen et al (3) and Marchalon i s e t al (ii) independently isolated and characterized the physical properties of Limulus agglutinin, including its serologic specificity (8). Androctonus australis (Saharan Scorpion), a terrestrial arthropod classified as a member of the class Arachnida and belonging to the order Scor~ioni4a, has been recorded in the northern fringes of the Sahara Desert of Algeria. It inhabits dwellings on burrows of desert regions and it is most active at night feeding on insects and spiders. 429

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ImmunoelectrophQresis of normal Androctonus hemolymph gives 13 arcs of precipitation: heavier arcs migrate toward the amode, lighter ones toward the cathode (2). Androctonus agglutinin is non-dialyzable, heat labile, and precipitates in distilled water, Brahmi and Cooper (1974). The present paper extends these observations and compares them with what is known about Limulus. MATERIALS AND METHODS Serum: Androctonus australis serum was separated from clotted hemolymph collected from living anZmals by Dr. Z. Brahmi, of the Institute Pasteur D'Algerie, Algiers, Algeria and individual sera pooled. The serum was shipped unfrozen, by air, to Buffalo, New York. Limulus polyphemus serum was obtained from the hemolymph of living specimens from Cape May Beach, Rutgers University, New Jersey. Erythrocytes: Whole blood from human donors was collected in disodium EDTA (9mg/7ml) and washed threee times with D.H.B. (Difco Hemagglutination Buffered Saline) pH 7.4 before use. Erythrocytes were prepared in 0.1% cell suspension for testing. Lymphocytes and Cell Lines: Heparinized samples were drawn from patients with chronic lymphocytic Leukemia. The lymphocytes were prepared by the method of Amos et al (i). Peripheral human lymphocytes were suspended in phosphate buffered saline, pH 7.4, at concentrations of ~aproximately 5,000 cells/cm3). The cultured lymphoblastoid cell lines of B-cells, B-1788 and of T-cells (Molt-4-F) were maintained in the laboratory using R.P.M.I. medium 1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C. They were grown at a logarithmic rate and the cell number increased exponentially. The B-1788 line was derived from peripheral blood lymphocytes of a healthy donor, and the Molt-4-F line was derived from leukemic blood of a patient with relapsed chronic lymphocytic leukemia (5). Sugars: Selected sugars were purchased from Sigma Chemical Company. Microagglutination Tests: Microagglutination tests were performed in small plastic trays (Falcon) for each of the respective cell lines. Serial two-fold dilutions of .005ml of both Limulus and Androctonus sera in buffered saline were placed in V-bottom microtiter plates to which were added equal volumes of the respective cell-suspensions. The cell-serum dilution mixtures were mixed immediately by horizontal plane agitation, then incubated at room temperature (22-25Oc) for 60 minutes. The tests were graded from 0-4 as to degrees of agglutinations; titers and scores were recorded. Hemagglutination Inhibition: Inhibition of hemagglutination of human erythrocytes by whole Androctonus and Limulus sera with selected sugars or sugar derivatives were investigated. Androctonus and Limulus sera were incubated separately at room temperature (22-25oc) with equimolar solutions (0.2M) of sugar or sugar derivatives for 30 minutes prior to microaaglutination tests (3).

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Neuraminidase Treatment: The neuraminidase (RDE) treatment of human erythrocytes was by the method of Neter et al (1959).

RESULTS Hemagglutination

of Human Er~throc~tes by Androctonus Australis Serum:

The titer of a serum is the reciprocal of the highest dilution showing microscopic agglutination. Androctonus serum gave a reciprocal of titer of 512 for specimens tested of each of the four human ABO blood groups. Summation of degrees of agglutination were 34, 28, 27 and 29, respectively, for the group O (H), A, B and AB erythrocyte suspensions tested. Further studies of hemagglutination of ABO (H) groups are in progress.

TABLE 1 Agglutination

of Peripheral & Cultured Human Lymphocytes by Androctonus & T.~mn]u~ Sera Score of Agglutination Range Median

Serum Androctonus Androctonus

Normal CLL

12 17

16-512 32-512

128 512

5-13 9-25

ii 20

Limulus Limulus

Normal CLL

8 12

2-64 2-512

8 32

5-11 4-20

7

Androctonus Androctonus

B(1788) T(Molt-4-F)

7 7

32-256 16-256

128 64

7-26 7-25

16 12

Limulus Limulus

B(1788) T(Molt-4-F)

6 6

4-256 16-256

64 64

8-16 9-15

13 12

NOTE:

Normal CLL B (1788) T(Molt-4-F)

= = = =

# of Subjects or Cell Line

Reciprocal of Titer Range Median

Cell Type

Normal Lymphocytes Chronic Leukemic Lymphocytes Cultured B Lymphoblastoid Cell Line Cultured T Lymphoblastoid Cell Line

8

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TABLE 2a The Score Frequency Distribution of De~rees of A @ @ l u t i n a t i o n for Peripheral and Cultured Lymphoblastoid Cell Lines for Androctonus and Limulu@ Sera

Androctonus Serum Scores 4 5 6 7 9 i0 ii 12 13 14 15 16 17 18 19 2O 21 22 23 24 25 26

Normal

CLL

B

T

1

Normal

CLL

B

T

1 1

1 3 2 3 2

1 1 5

1

1 3 1

3 2 1 1

1 1 2 1 1 2

1

1

1 1 1 1 1

3

1

2 2 1

2

1

2

3 1 B

Total

12

17

7

7

8

12

Sample Variance 6.20

32.63

47.91 68.83

3.14

21.41

Mean

10.25

16.00

14.28 18.14

8.50

11.16

.72

1.39

.63

1.34

S.E.M. NOTE:

2.62

3.14

See Table 1 for abbreviations under cells

6

5.52

6

5.50

11.83 12.50 .96

.96

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2

The Titer Frequency Distribution of Degrees of Ag@lutination for Peripheral and C u l t u r e d L~m~hoblastoid Cell Lines for Androctonus and Limulus Sera

Serum

Serum

L°g2 1 Titer2 3 4 5 6 7 8 9 Total

Titer-i Normal

E x p r e s s e d as Log 2 CLL B T

4 3 1 3 1 3

1 3 i0

12

17

1 3 2 1

1 1 2

Titer -I Normal 1 2 2 1 2

Expressed as Log 2 CLL B T 1 3 5 1

3

1 2 2 1 1 1

2 2

6

6

2 7

7

8

12

Sample Variance 4.27

2.38

0.95

2.58

2.13

4.25

4.30

3.51

Mean

6.50

8.00

6.42

6.42

4.12

5.33

5.50

6.33

.60

.37

.37

.60

.53

.60

.85

.76

S.E.M. NOTE:

Titer -I = Reciprocal of titer S.E.M. = Standard error of mean See Table 1 for abbreviations under cells

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TABLE 3 Pairwise Comparison by t-Test of A g g l u t i n a t i o n of Peripheral and Cultured Lymphocytes by Androctonus Serum Differences Between Log 2 Titer -I Cells

Differences

CLL-Normal 1.5 CLL-B 1.58 CLL-T 1.58 B-Normal -0.08 T-Normal 0.08 B-T 0.00 * ** ***

STE

T

.70 .52 .71 .70 .85 .71

2.13" 3.02** 2.22** .ii .09 .00

Differences Between Mean Scores Differences 5.75 .0 -2.14 5.75 7.89 -3.86

S-E 1.56 3.04 3.43 2.80 3.22 4.07

T 3.69*** .59 .62 2.05* 2.45* .95

Significant at .05 level Significant at .01 level Significant at .001 level

NOTE:

See Table 1 for abbreviations under cells

TABLE 3a Pairwise Comparison by t-Test of A ~ g l u t i n a t i o n of Peripheral and Cultured Lymphocytes by Limulus Serum Differences Between Log2 Titer -I Cells

Differences

CLL-Normal 1.13 CLL-B -0.25 CLL-T -1.08 B-Normal 1.38 T-Normal 2.21 B-T -0.83 * **

S-E .80 1.03 .97 1.00 0.93 1.14

T 1.42 0.22 1.12 1.38 2.38* 0.70

Differences Between Mean Scores Differences 2.66 -6.77 -1.34 3.33 4.00 -.67

Significant at 0.05 level Significant at 0.01 level

NOTE:

See Table 1 for abbreviations under cells

S-E 1.70 1.30 1.30 1.45 1.45 1.35

T

1.81 0.42 0.82 2.29* 2.76** .49

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Summation of results of Androctonus and Limulus sera tested with different lymphocytes is shown in Table i. Human peripheral chronic lymphocytic leukemic lymphocytes (CLL) were agglutinated by Androctonus australis serum to a higher titer than were normal human peripheral lymphocytes. Median reciprocals of titers were 512 for the CLL cells and 128 for the normal cells, ranges were 32-512 and 16-512 for CLL and normal cells. Titer and score differences were definitely observed, as well as prozone phenomena. A n d r o c t o n u s serum gave higher titers than did Limulus serum tested with the same CLL and normal cells. Limulus s e r u m could differentiate between the CLL and normal cells confirming the previous work by Cohen et al (5). The titer and score differences were not considered significant between the agglutinations of B and T cells with either Androctonus or Limulus sera. The frequency distributions for each of the four cell types for both Androctonus and Limulus are shown in Table 2 and 2a. Higher mean titers were obtained from normal lymphocytes or cultured T and B lymphoblastoid cells, each respectively tested w i t h Androctonus serum. Androctonus serum also showed higher mean scores for peripheral CLL, than for normal lymphocytes. W h e n Androctonus serum was tested with cultured T and B lymphoblastoid cell lines, higher mean scores were obtained for B-cells than CLL cells. Limulus polyphemus serum showed higher mean titers and scores for peripheral CLL rather than normal lymphocytes. The mean titer and scores by Limulus serum for the peripheral CLL were lower than those for T and B lymphoblastoid cell lines. Tables 3 and 3a present t-test analyses for the pairwise differences found between the mean values for log titers and scores for both Androctonus and Limulus sera. In Table 3, statistically significant differences are demonstrated: for the mean titers and scores between Androctonus agglutination of CLL and normal lymphocytes, for the mean titers b e t w e e n CLL and B cells and for the m e a n titers between CLL and T cells. The mean agglutination scores between T and normal cells and between B and normal cells are statistically significant. In addition, statistically significant differences were demonstrated between m e a n scores for T and B cells versus normal cells, and between the mean titers of T and normal cells tested w i t h Limulus serum. B and T cultured cells, when tested w i t h Limulus or with An~roctonus sera, showed no significant differences. Comparisions based on titers were not always consistent w i t h comparisons based on scores (Tables 3 and 3a).

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TABLE 4 Effect of Neur...aminidase Treatment on H e m a ~ g l u t i n a t i o n Tested with Human Erythrocytes Androctonus Serum Untreated Treated Titer -1

256 256 256 256 NOTE:

Score

28 28 27 19

Titer -1

Tested with Human Erythrocytes Limulus Serum - Lot #61-3297 Untreated Treated

Score

256 256 256 128

Titer -1

Score

Titer -1

Score

16 16 16 32

6 9 6 7

8 4 8 2

8 5 4 3

27 24 19 21

Titer -I'= Reciprocal of Titer Score = Sun~ation of A g g l u t i n a t i o n

Human erythrocytes treated with neuraminidase gave minimal to negative agglutination with Limulus serum (Table 4) but the similarly treated cells gave much higher titers and scores w i t h Androctonus serum. The g a m e erythrocyte donor was used each time for the titrations of Androctonus and Limulus sera.

TABLE 5 Comparison of Inhibition of Hemag~lutination of Human Erythrocytes b~ Whole Androctonus and Limulus Sera Tested with Selected Sugars or Sugar Derivative Inhibitor Sugar Derivative (0.2M)

Androctonus Serum Titer -I

Score

Limulus Serum Lot #61-3297 Titer -I Score

N-Acetyl-D-Galactosamine

256 128 128

14 19 14

4 4 2

3 3 2.5

N-Acetyl-D-Glucosamine

256 64 64

16 17 9

8 4 2

4.5 3.5 2.0

N-Acetyl-Neuraminic Acid

16 N.T N.T

4 N.T N.T

2 UN 2

i. 5 1.0 1.5

64 14 128 128 17 64 256 16 8 Score = Su~nation of degrees of agglutination Titer -I = Reciprocal of Titer, UN = Undiluted,

ii 12 4.5

Saline Control

NOTE:

N.T = Not Tested

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The activity of Limulus hemolymph was inhibited by N-acetyl-Dgalactosamine, N-acetyl-D-glucosamine and N-acetyl-neuraminic acid (Table 5). There were slight inhibitory effects by the selected sugars or sugar derivatives tested with Androctonus hemolymph, with marked inhibition by N-acetylneuraminic acid and a drop in the agglutination scores. DISCUSSION The serologic specificities of agglutinins of Androctonus australis have been compared with agglutinins of Limulus polyphemus. Several possible explanations, from the data presented in Tables 1 to 5, will now be considered. Androctonus australis serum was able to differentiate between human peripheral lymphocytes and human peripheral leukemic lymphocytes (CLL), possibly due to the presence of inhibitory or non-inhibitory molecules on the receptors. The hypothesis just presented appears to be in accord with the observations of Race et al (15) and Neter et al (12). Limulus polyphemus serum differentiated between normal human peripheral lymphocytes and human peripheral leukemic lymphocytes. Molt-4-F and B-1788 were obtained from cultures in the same exponential growth phase. For this reason, the agglutination of the cells would reflect differences in cell surface properties rather than differences due to cell growth, but this was not the case. It is not apparent from these experiments whether the B/T cell ratios are responsible for the differentiation of leukemic from normal lymphocytes as reported earlier, with Limulus serum (5). Although both Androctonus and Limulus sera were able to differentiate the CLL from normal lymphocytes, Androctonus nonetheless showed greated degrees of agglutination. Relative agglutination titers suggest differences in the receptor sites for Androctonus and for Limulus agglutinins. Androctonus serum agglutinated the human erythrocytes to higher titers and scores and with no significant differences noted in the ABO types. Serum from Androctonus australis is capable of agglutinating erythrocytes from a variety of vertebrate species (2). Limulus, by contrast, agglutinated human erythrocytes with differentially greater specificity for the human A and B erythrocyte antigens (3). The differences observed with human erythrocyte antigens for Androctonus and Limulus agglutinins are not readily explained on this basis. However, one speculation may be that there are differences in sizes, shapes and capacities for interactions with receptors for the two agglutinins, i.e., different association constants. With regard to the effects of neuraminidase treatment on hemagglutination, Cohen (1968), first reported the inhibition of agglutination of human erythrocytes by Limulus agglutinin by a saccharide derivative N-acetyl-D-Glucosamine (NADG), suggesting the importance of sialic acid as a cell specific receptor for the agglutinin. This was confirmed by Pardoe et al (24). As a consequence of the inhibitory effect of NADG, the effects of neuraminidase treatment of

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human erythrocytes on agglutination of Androctonus were compared with those of Limulus agglutinins. Enzyme treated cells, tested in the Androctonus serum showed evidence of agglutination, suggesting that neuraminidase treatment does not eliminate agglutination. This means that the receptor sites on the human erythrocytes are accessible to the Androctonus agglutinin molecules irrespective of neuraminidase treatment or else other receptors are important. Experiments show that hemagglutination by Androctonus hemolymph is inhibited by N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and most markedly, by N-acetyl-neuraminic acid. Androctonus agglutinins appear to have a different affinity and different specificity for inhibitor sugar derivatives than do Limulus agglutinins. The derivatives N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and N-acetyl-neuraminic acid inhibit the agglutination of human erythrocytes by Limulus ~olyphemus serum. Such results with Limulus polyphemus are similar to those reported by Satoru et al (16), that N-Acetyl-Dgalactosamine, N-acetyl-neuraminic acid, N-acetyl-D-glucosamine inhibit the activity of Tachypleus tridentatus (Japanese Horse-shoe crab). The cell receptor site for Limulus agglutinin has been part of a concept of cell membranes proposed by Uhlenbruck (18 and 19). Cohen et al (8), utilized Limulus agglutinins in the study of the nature of human blood group MN determinants which suggested that N-acetyl-neuraminyl (NANA) receptor was common to both M and N determinants. In summary, the results in this report suggest that Androctonus and Limulus sera may be of use not only for identification of leukemic or "neoplastic" human cells, but also in studies of topographic changes in normal and leukemic lymphocyte cell membranes. We hope that the biomedical applications of Androctonus and Limulus cellular agglutinins will be investigated further in the same manner as other invertebrate agglutinins (4,9, i0 and 17).

ACKNOWLEDGEMENTS Support is acknowledged of the Juliette and Israel Cohen Memorial Research Fund. Grateful acknowledgements are made to Ms. S.G. Gregory for her technical assistance, Mrs. V. Belert, R.N., who procured blood samples from chronic lymphocytic leukemic patients and Plasmapheresis Donor Room nurses, Mrs. M.S. Pfeil, R.N. and Mrs. Bea McGaw, R.N., who obtained normal blood samples. Professors Alan A. Boyden and Mabel Boyden of Rutgers University personally collected Limulus hemolymph for these studies. Theodore W. Ritter, Williamsville East High School, screened pools of Limulus sera for those with hemagglutinins that gave high titers and scores. The experiments were performed in laboratories of the Department of Medicine, Dr. John E. Fitzpatrick, Chief. We acknowledge the helpful criticisms and suggestions manuscript made by Professor Edwin L. Cooper.

for improving the

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