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Agglutinin-mediated attachment of erythrocytes to hemocytes of Helix pomatia
JOURNAL
OF INVERTEBRATE
PATHOLOGY
29,97- 100(1977)
Agglutinin-Mediated Attachment of Erythrocytes Hemocytes of Helix pomatial
to
LOTHAR R. RENWRANTZ~ANDTHOMASC.CHENG Institute for Pathobiology, Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015 Received September 16, 1976 In order to ascertain whether agglutinins can serve as links to bind hemocytes of Helix pomatia to mammalian erythrocytes, rosette-formation tests were performed. These involved pretreatment of H. pomatia hemocytes with each of 15 nonnative agglutinins and incubation of them with human erythrocytes. It has been found that. of the agglutinins tested, wheat germ agglutinin (WGA) as well as those from Ricks communis, Axinella polypoides, Anguilla anguilla (anti-H,,,), concanavalin A, and Limulus polyphemus caused rosette formation with human erythrocytes. In addition, it has been found that a small number of H. pomatia hemocytes are capable of direct binding to erythrocytes of mice, rabbits, rats, and sheep.
procedures followed in maintaining, bleeding, and isolating their hemocytes were presented earlier (Renwrantz and Cheng, 1977). In order to demonstrate whether exogenous agglutinins will bind mammalian erythrocytes to hemocytes of H. pomatia, rosette-formation tests were performed. Rosette-formation tests in tubes. Freshly collected and isolated hemocytes of H. pomatia were suspended in isotonic saline (4-6 x lo6 cells/ml) in a molluscan Ringer’s solution, as described by Roach (1963), and in the same molluscan Ringer’s solution fortified with 10% heat-inactivated calf serum (Grand Island Biological Co., Grand Island, New York). The mammalian erythrocytes employed in this series of tests were in the form of 0.5% suspensions of cells of the human A, B, and 0 types and cells of guinea pigs, mice, rabbits, rats. and sheep in a 200 mo.sM NaCl solution. Samples, each consisting of 150 ~1 of the various suspensions of erythrocytes, were mixed in polyethylene tubes with an identical amount of the Helix hemocyte suspensions. The preparations were then centrifuged for 10 min at 4OOg and stored for 1 hr at 4°C. Alternatively, the prepara-
INTRODUCTION
In an earlier publication (Renwrantz and Cheng, 1977) it was reported that specific binding sites occur on the surface of hemocytes of the pulmonate gastropod Helix porn&z. Furthermore, it has been reported that these receptor sites are comprised in part of specific carbohydrates. It was announced in the earlier report that we conducted studies to ascertain whether aggiutinins can function as links to join foreign particles, e.g., vertebrate erythrocytes, to hemocytes of H. porn&a. The results of these studies are reported herein. MATERIALS
AND METHODS
The specimens of H. pomatia employed in our studies were purchased from Robert Stein Co., Lauingen, West Germany. The ’ This research was supported by a grant (AI 1235502-Al) from the National Institute of Allergy and Infectious Diseases and a grant (FD 00416-06) from the Food and Drug Administration. U.S. Public Health Service. ’ Visiting Research Scientist from the Zoologisches Institut und Zoologisches Museum, Universitat Hamburg, Germany, sponsored by the Deutsche Forschungsgemeinschaft. 97 Copyright All right\
0 1977 by Academic Press. Inc. of reproduction m any form reserved.
98
RENWRANTZ
tions were stored overnight at 4°C to permit the cells to settle. In either case, the pellets were gently resuspended and samples were prepared on slides for microscopical examination. The number of Helix hemocytes binding three or more erythrocytes was ascertained. Rosette-formation test of hemocytes pretreated with agglutinins. Two drops of fresh hemolymph were placed on each glass slide and left undisturbed for 10 min at room temperature (22°C). The serum was subsequently washed off with a gentle stream of isotonic saline, and the monolayers of cells were overlaid with the following agglutinins: anti-A,, (an extract of Dolichos biflorus seeds); anti-hH (an extract of the albumin gland of Cepaea hortensis); antiACN (an extract of the albumin gland of Cepaea nemoralis); anti-A,, (an extract of the albumin gland of Helix pomatia); the agglutinins extracted from the seeds of Soja sp., Ricinus communis, and Uiex europaeus; those extracted from the poriferan Axinella polypoides and from the coelenterate Cerianthus sp.; anti-H,,, (the agglutinin in the serum of the eel, Anguilla anguilla); and the agglutinin in the serum of Limulus polyphemus. All of the extractions were performed by the method of Renwrantz et al. (1975). In addition, several commercially available agglutinins were also tested. These were Laburnum alpinum agglutinin (Behringwerke Co., Marburg-Lahn, Germany), phytohemagglutinin, M form (PHA) (Grand Island Biological Co., Grand Island, New York), the wheat germ agglutinin (WGA) (Serva Feinbiochemica, Heidelberg, Germany), and concanavalin A (Con A) (Calbiochem Co., LaJolla, California). All of the agglutinin solutions were prepared in a 200 mosM NaCL solution. The Helix hemocyte-agglutinin preparations were stored in a moisture chamber for approximately 1 hr at room temperature. Subsequently, the nonbound agglutinins were rinsed off with isotonic saline, and the preparations were covered with hu-
AND
CHENG
man erythrocyte suspensions for a minimum of 45 min at room temperature. After the unbound erythrocytes were removed by rinsing, the monolayer preparations were covered with saline and a coverglass and examined microscopically for binding of erythrocytes. Again, the finding of three or more human erythrocytes bound to a Helix hemocyte was considered a true rosette formation. RESULTS
Approximately 700 molluscan cells of each of the various Helix hemocyte-erythrocyte mixtures in NaCl solution were examined to quantify a direct binding of Helix hemocytes to mammalian red cells. No rosette formation was observed with guinea pig and human ABO erythrocytes, but about 3% of the hemocytes examined served as the center for the formation of rosettes by erythrocytes of mice (2.5%), rabbits (2.7%). and rats (4%), while 8% of the Helix cells bound three or more sheep red blood cells. To determine an influence of the suspension medium on rosetting, hemocytes, and erythrocytes were mixed in two additional solutions (see Materials and Methods). In both cases, there was no increase in the percentage of rosettes formed. Rosette formation of pretreated hemocytes. The results of our rosette-formation
FIG. rocytes pomatia
1. Rosette formation with wheat germ hemocytes.
of human 0 type agglutinin-coated
erythHe/ix
ATTACHMENT
TO HELIX
tests on slides involving Helix hemocytes pretreated with agglutinins are tabulated in Table 1. It is noted that the human erythrocytes formed rosettes with Helix cells that had been coated with six of the agglutinins tested: WGA, Ricinus, Axinella, anti-H,,, , Con A. and Limulus agglutinins. Most of these hemocytes were bound to more than three human erythrocytes, with some completely surrounded by erythrocytes (Figs. 1, 2). No attempt was made to quantify the number of agglutinincoated Helix hemocytes that had formed rosettes with human erythrocytes since we could not be certain that some of the rosettes had not been destroyed during the washing process. Nevertheless, the number of pretreated molluscan hemocytes that had formed rosettes with human erythrocytes consistently exceeded 60%.
HEMOCYTES
99
FIG. 2. Rosette formation of human 0 type erythrocytes with Con A-coated Helix pomatia hemocytes.
the surface of the hemocytes of H. pomatin. Now, as a result of our investigations presented herein, it is evident that certain agglutinins can serve as connecting bridges between the surface receptors of Helix TABLE 1 hemocytes and human erythrocytes. It should be emphasized, however, that the ROSETTE FORMATION OF HUMAN ERYTHROCYTES absence of rosette formation by human erythWITH AGGLUTININ-PRETREATED HELIX HEMOCYTES rocytes with Helix hemocytes that had Rosette been incubated with an agglutinin need not Agglutinins Erythrocytes” formation* mean that receptor sites are absent from the molluscan cells. The “incompleteness” Anti-AD,, A of some agglutinins (Prokop et al., 1968). or Anti-AuH A A Anti-A,, inappropriate methodology, may be a reaA Anti-AHP son for the failure to form rosettes. Soja 0 It has been reported that there is some PHA 0 direct attachment of erythrocytes of mice, + WGA 0 rabbits, rats, and sheep to noncoated Helix Ricinus 0 + Axinella 0 + cells. A similar observation has been reCerianthus 0 ported by McKay and Jenkin (1970) who Anti-H,,, 0 + found that a small percentage of hemoLaburnum 0 cytes of crayfish will bind directly to sheep Ulex 0 erythrocytes. In addition, Cooper (1973) Con A 0 + Limrrlrrs 0 + has reported the direct attachment of sheep erythrocytes to 15-17% of washed earth” The letters designate the type of human erythroworm coelomocytes. Thus, there is some cytes. evidence that certain cells of inverte* - = no rosettes, + = rosette formation. brates, including H. pornatia, are capable of direct binding to certain types of mamDISCUSSION AND CONCLUSIONS malian erythrocytes. In the earlier report (Renwrantz and In conclusion, it is now apparent that an Cheng, 1977), we documented the finding of attachment of erythrocytes to the surface receptor sites for specific agglutinins on of hemocytes of H. pomatia may be caused
100
RENWRANTZ
by agglutinins which serve to link nonself material to the phagocyte; however, small numbers of Helix hemocytes are also capable of direct binding of certain foreign cells. REFERENCES E. W. 1973. Evolution of cellular immunity. In “Nonspecific Factors Influencing Host Resistance,” (W. Braun and J. Ungar, eds.). pp. 1l-23. Karger, Basel. MCKAY, D., AND JENKIN. C. R. 1970. Immunity in the invertebrates. The role of serum factors in phagocytosis of erythrocytes by haemocytes of COOPER,
AND CHENG the freshwater crayfish (Purachaerups bicarinatus). Aust. J. Exp. Biol. Med. Sri., 48, 139-150. PROKOP, O., UHLENBRUCK, G., AND K~HLER, W. 1968. A new source of antibody-like substances having anti-blood group specificity. VOX Sang., 14, 321-333. RENWRANTZ. L., BRETTING, H., SALFNER, J., AND UHLENBRUCK. G. 1975. Uber das Vorkommen von kohlenhydrathaltigen PrSzipitinogenen in marinen Invertebraten und Eiern einiger Fischarten. Biol. Zentralbl., 94, 431-440. RENWRANTZ, L., AND CHENG, T. C. 1977. Identification of agglutinin receptors on hemocytes of Helix pomatia. J. Invertebr. Pathol., 29, 88-%. ROACH, D. K. 1%3. Analysis of the hemolymph of Arion ater L. (Gastropoda, Rulmonata). J. Exp. Biol., 40, 613-623.
OF INVERTEBRATE
PATHOLOGY
29,97- 100(1977)
Agglutinin-Mediated Attachment of Erythrocytes Hemocytes of Helix pomatial
to
LOTHAR R. RENWRANTZ~ANDTHOMASC.CHENG Institute for Pathobiology, Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015 Received September 16, 1976 In order to ascertain whether agglutinins can serve as links to bind hemocytes of Helix pomatia to mammalian erythrocytes, rosette-formation tests were performed. These involved pretreatment of H. pomatia hemocytes with each of 15 nonnative agglutinins and incubation of them with human erythrocytes. It has been found that. of the agglutinins tested, wheat germ agglutinin (WGA) as well as those from Ricks communis, Axinella polypoides, Anguilla anguilla (anti-H,,,), concanavalin A, and Limulus polyphemus caused rosette formation with human erythrocytes. In addition, it has been found that a small number of H. pomatia hemocytes are capable of direct binding to erythrocytes of mice, rabbits, rats, and sheep.
procedures followed in maintaining, bleeding, and isolating their hemocytes were presented earlier (Renwrantz and Cheng, 1977). In order to demonstrate whether exogenous agglutinins will bind mammalian erythrocytes to hemocytes of H. pomatia, rosette-formation tests were performed. Rosette-formation tests in tubes. Freshly collected and isolated hemocytes of H. pomatia were suspended in isotonic saline (4-6 x lo6 cells/ml) in a molluscan Ringer’s solution, as described by Roach (1963), and in the same molluscan Ringer’s solution fortified with 10% heat-inactivated calf serum (Grand Island Biological Co., Grand Island, New York). The mammalian erythrocytes employed in this series of tests were in the form of 0.5% suspensions of cells of the human A, B, and 0 types and cells of guinea pigs, mice, rabbits, rats. and sheep in a 200 mo.sM NaCl solution. Samples, each consisting of 150 ~1 of the various suspensions of erythrocytes, were mixed in polyethylene tubes with an identical amount of the Helix hemocyte suspensions. The preparations were then centrifuged for 10 min at 4OOg and stored for 1 hr at 4°C. Alternatively, the prepara-
INTRODUCTION
In an earlier publication (Renwrantz and Cheng, 1977) it was reported that specific binding sites occur on the surface of hemocytes of the pulmonate gastropod Helix porn&z. Furthermore, it has been reported that these receptor sites are comprised in part of specific carbohydrates. It was announced in the earlier report that we conducted studies to ascertain whether aggiutinins can function as links to join foreign particles, e.g., vertebrate erythrocytes, to hemocytes of H. porn&a. The results of these studies are reported herein. MATERIALS
AND METHODS
The specimens of H. pomatia employed in our studies were purchased from Robert Stein Co., Lauingen, West Germany. The ’ This research was supported by a grant (AI 1235502-Al) from the National Institute of Allergy and Infectious Diseases and a grant (FD 00416-06) from the Food and Drug Administration. U.S. Public Health Service. ’ Visiting Research Scientist from the Zoologisches Institut und Zoologisches Museum, Universitat Hamburg, Germany, sponsored by the Deutsche Forschungsgemeinschaft. 97 Copyright All right\
0 1977 by Academic Press. Inc. of reproduction m any form reserved.
98
RENWRANTZ
tions were stored overnight at 4°C to permit the cells to settle. In either case, the pellets were gently resuspended and samples were prepared on slides for microscopical examination. The number of Helix hemocytes binding three or more erythrocytes was ascertained. Rosette-formation test of hemocytes pretreated with agglutinins. Two drops of fresh hemolymph were placed on each glass slide and left undisturbed for 10 min at room temperature (22°C). The serum was subsequently washed off with a gentle stream of isotonic saline, and the monolayers of cells were overlaid with the following agglutinins: anti-A,, (an extract of Dolichos biflorus seeds); anti-hH (an extract of the albumin gland of Cepaea hortensis); antiACN (an extract of the albumin gland of Cepaea nemoralis); anti-A,, (an extract of the albumin gland of Helix pomatia); the agglutinins extracted from the seeds of Soja sp., Ricinus communis, and Uiex europaeus; those extracted from the poriferan Axinella polypoides and from the coelenterate Cerianthus sp.; anti-H,,, (the agglutinin in the serum of the eel, Anguilla anguilla); and the agglutinin in the serum of Limulus polyphemus. All of the extractions were performed by the method of Renwrantz et al. (1975). In addition, several commercially available agglutinins were also tested. These were Laburnum alpinum agglutinin (Behringwerke Co., Marburg-Lahn, Germany), phytohemagglutinin, M form (PHA) (Grand Island Biological Co., Grand Island, New York), the wheat germ agglutinin (WGA) (Serva Feinbiochemica, Heidelberg, Germany), and concanavalin A (Con A) (Calbiochem Co., LaJolla, California). All of the agglutinin solutions were prepared in a 200 mosM NaCL solution. The Helix hemocyte-agglutinin preparations were stored in a moisture chamber for approximately 1 hr at room temperature. Subsequently, the nonbound agglutinins were rinsed off with isotonic saline, and the preparations were covered with hu-
AND
CHENG
man erythrocyte suspensions for a minimum of 45 min at room temperature. After the unbound erythrocytes were removed by rinsing, the monolayer preparations were covered with saline and a coverglass and examined microscopically for binding of erythrocytes. Again, the finding of three or more human erythrocytes bound to a Helix hemocyte was considered a true rosette formation. RESULTS
Approximately 700 molluscan cells of each of the various Helix hemocyte-erythrocyte mixtures in NaCl solution were examined to quantify a direct binding of Helix hemocytes to mammalian red cells. No rosette formation was observed with guinea pig and human ABO erythrocytes, but about 3% of the hemocytes examined served as the center for the formation of rosettes by erythrocytes of mice (2.5%), rabbits (2.7%). and rats (4%), while 8% of the Helix cells bound three or more sheep red blood cells. To determine an influence of the suspension medium on rosetting, hemocytes, and erythrocytes were mixed in two additional solutions (see Materials and Methods). In both cases, there was no increase in the percentage of rosettes formed. Rosette formation of pretreated hemocytes. The results of our rosette-formation
FIG. rocytes pomatia
1. Rosette formation with wheat germ hemocytes.
of human 0 type agglutinin-coated
erythHe/ix
ATTACHMENT
TO HELIX
tests on slides involving Helix hemocytes pretreated with agglutinins are tabulated in Table 1. It is noted that the human erythrocytes formed rosettes with Helix cells that had been coated with six of the agglutinins tested: WGA, Ricinus, Axinella, anti-H,,, , Con A. and Limulus agglutinins. Most of these hemocytes were bound to more than three human erythrocytes, with some completely surrounded by erythrocytes (Figs. 1, 2). No attempt was made to quantify the number of agglutinincoated Helix hemocytes that had formed rosettes with human erythrocytes since we could not be certain that some of the rosettes had not been destroyed during the washing process. Nevertheless, the number of pretreated molluscan hemocytes that had formed rosettes with human erythrocytes consistently exceeded 60%.
HEMOCYTES
99
FIG. 2. Rosette formation of human 0 type erythrocytes with Con A-coated Helix pomatia hemocytes.
the surface of the hemocytes of H. pomatin. Now, as a result of our investigations presented herein, it is evident that certain agglutinins can serve as connecting bridges between the surface receptors of Helix TABLE 1 hemocytes and human erythrocytes. It should be emphasized, however, that the ROSETTE FORMATION OF HUMAN ERYTHROCYTES absence of rosette formation by human erythWITH AGGLUTININ-PRETREATED HELIX HEMOCYTES rocytes with Helix hemocytes that had Rosette been incubated with an agglutinin need not Agglutinins Erythrocytes” formation* mean that receptor sites are absent from the molluscan cells. The “incompleteness” Anti-AD,, A of some agglutinins (Prokop et al., 1968). or Anti-AuH A A Anti-A,, inappropriate methodology, may be a reaA Anti-AHP son for the failure to form rosettes. Soja 0 It has been reported that there is some PHA 0 direct attachment of erythrocytes of mice, + WGA 0 rabbits, rats, and sheep to noncoated Helix Ricinus 0 + Axinella 0 + cells. A similar observation has been reCerianthus 0 ported by McKay and Jenkin (1970) who Anti-H,,, 0 + found that a small percentage of hemoLaburnum 0 cytes of crayfish will bind directly to sheep Ulex 0 erythrocytes. In addition, Cooper (1973) Con A 0 + Limrrlrrs 0 + has reported the direct attachment of sheep erythrocytes to 15-17% of washed earth” The letters designate the type of human erythroworm coelomocytes. Thus, there is some cytes. evidence that certain cells of inverte* - = no rosettes, + = rosette formation. brates, including H. pornatia, are capable of direct binding to certain types of mamDISCUSSION AND CONCLUSIONS malian erythrocytes. In the earlier report (Renwrantz and In conclusion, it is now apparent that an Cheng, 1977), we documented the finding of attachment of erythrocytes to the surface receptor sites for specific agglutinins on of hemocytes of H. pomatia may be caused
100
RENWRANTZ
by agglutinins which serve to link nonself material to the phagocyte; however, small numbers of Helix hemocytes are also capable of direct binding of certain foreign cells. REFERENCES E. W. 1973. Evolution of cellular immunity. In “Nonspecific Factors Influencing Host Resistance,” (W. Braun and J. Ungar, eds.). pp. 1l-23. Karger, Basel. MCKAY, D., AND JENKIN. C. R. 1970. Immunity in the invertebrates. The role of serum factors in phagocytosis of erythrocytes by haemocytes of COOPER,
AND CHENG the freshwater crayfish (Purachaerups bicarinatus). Aust. J. Exp. Biol. Med. Sri., 48, 139-150. PROKOP, O., UHLENBRUCK, G., AND K~HLER, W. 1968. A new source of antibody-like substances having anti-blood group specificity. VOX Sang., 14, 321-333. RENWRANTZ. L., BRETTING, H., SALFNER, J., AND UHLENBRUCK. G. 1975. Uber das Vorkommen von kohlenhydrathaltigen PrSzipitinogenen in marinen Invertebraten und Eiern einiger Fischarten. Biol. Zentralbl., 94, 431-440. RENWRANTZ, L., AND CHENG, T. C. 1977. Identification of agglutinin receptors on hemocytes of Helix pomatia. J. Invertebr. Pathol., 29, 88-%. ROACH, D. K. 1%3. Analysis of the hemolymph of Arion ater L. (Gastropoda, Rulmonata). J. Exp. Biol., 40, 613-623.