|Role of carcinoscorpin, a haemolymph lectin of horseshoe crab Carcinoscorpius rotundacauda as humoral factor|
S Basu Sarbadhikari*, D Datta and R Bhadra
Department of Cellular Biochemistry, Indian
Institute of Chemical Biology, 4, Raja S C
Calcutta 700 032, India
*Indian Association for the Cultivation of Science, 5, Raja S C Mullick Road, Calcutta 700 032, India
Corresponding author (Fax, 91-33-4730284; Email, firstname.lastname@example.org).
This study demonstrated that a marine Indian horseshoe crab, Carcinoscorpious rotundacauda showed higher self defence in an experimental infection upon the induction of its circulatory lectin, carcinoscorpin. It resisted an infection with 107 live Escherichia coli per crab when the circulatory carcinoscorpin was 816-fold higher after administering 2-ketodeoxyoctonate (Kdo) into the live crab. The naive control with its natural level of circulatory lectin could tolerate a maximum infective dose of 106 live E. coli per crab. Bacterial killing and phagocytic uptake in association with the isolated crab amoebocytes in an ex vivo system was considerably higher for the lectin opsonized E. coli compared to unopsonized samples. Carcinoscorpin is thus functionally an opsonin in the defence of the primitive marine arthropod, C. rotundacauda, like vertebrate antibody, a humoral factor involved in the defence of the host. The natural capacity for defending an infection with 106 live E. coli per crab suggested that the crabs in the natural habitat hardly face such an infection and is possibly one of the reasons for its survival over millions of years as a living fossil.
The underlying defence system enabling the horseshoe crab, a marine arthropod to survive millions of years as an evolutionarily important "living fossil" (Pistole and Britko 1978) remained poorly understood. It is of considerable scientific interest to know the immune system of this arthropod. Possibly it would help to understand the evolution of immunity (Marchalonis and Schluter 1989) and provide the basis of immune response in an invertebrate that survived for millions of years (Christensen and Nappi 1988).
An agglutinin was discovered in the haemolymph of the horseshoe crab, Limulus polyphemus, a primitive marine arthropod. But immunoglobulins (Igs) like molecules of the vertebrates have not been detected in the circula-tion (Lackie 1988). The agglutinin subsequently characterized as a sialic acid, a saccharide acid binding protein or lectin (Goldstein et al 1980) and called limulin, was proposed to play some role on the host defense (Finstad et al 1974).
In higher vertebrate binding of the protective antibody to the bacterial pathogen causes its inactivation and helps for its phagocytosis to clear from the circulation of the host. Lectin mediated phagocytosis of bacteria by vertebrate macrophages has long been proposed as the basis of non specific host defence in the absence of antibody (Sharon 1984). Like the production of vertebrate antibody by immunization with antigen, the induction of the invertebrate circulatory lectin was first demonstrated by administering lectin specific haptenic carbohydrates of bacterial cell wall constituent, 2-ketodeoxyoctonate (Kdo) and b -glycerophosphate in Carcinoscorpius rotundacauda, an Indian horseshoe crab (Basu Sarbadhikari 1989; Basu Sarbadhikari and Bhadra 1990). However it was not known that whether such induced lectin would play any role on the host defence like the vertebrate antibody. Enhanced clearance of an invading microorganism by lectin opsonization was reported in the invertebrate system (Renwrantz and Stahmer 1983; Brehelin 1986; Fryer et al 1989). However enhanced host defence corresponding to the level of induced circulatory lectin is not known in invertebrate like vertebrate immune system involving antibodies and memory (Berzofsky 1985; Germain 1986; Berzofsky et al 1988).
Fatal intravascular clot formation leading to death occurred after the administration of Gram-negative Escherichia coli into live L. polyphemus and lipopolysaccharide (LPS) of this Gram-negative bacteria induced the clot after interaction with crab amoebocytes. Like Limulus, the amoebocytes of C. rotundacauda were shown to be equally sensitive to bacterial LPS for producing clot or gel (Mahalanobis et al 1979). Therefore, taking E. coli as an apparent pathogen of C. rotundacauda this study was undertaken to see the role of lectin in the host defence.
This study demonstrated that the circulatory carcinoscorpin acted as an opsonizing agent like vertebrate antibody, provided higher host resistance for an induced circulatory level and enhanced rate of phagocytosis for lectin opsonized bacteria thus playing the role of a humoral factor of vertebrate.
2. Materials and methods
Kdo was purchased from Sigma Chemicals Co. (USA) and used as an antigen like agent for induction of the circulatory lectin in live horseshoe crab, C. rotundacauda.
The collection of the horseshoe crabs, their maintenance in the laboratory, checking of Kdo for LPS contamination, lectin induction after the administration of Kdo, and selection of the crabs having 816 times higher circulatory level of carcinoscorpin were all carried out as described in our earlier study (Basu Sarbadhikari and Bhadra 1990). In each experiment, three groups, each consisting of 57 crabs as experimental organism were used for the administration of Kdo solution (1 mg/ml) to induce circulatory lectin, together with 3 groups (control) consisting of 34 crabs injected with pyrogen free 3% NaCl solution. Each experiment was repeated three times during different months of the year. The crabs used were adults, weighing 8001200 g each.
2.1 Preparation of carcinoscorpin and its antisera
The level of induced carcinoscorpin was determined by haemagglutination, a generalized technique used for the assay of all lectins, and it was necessary to authenticate the induced lectin as carcinoscorpin. Incubation of carcinoscorpin with its antisera for 3 h abolished the lectin activity totally (Basu Sarbadhikari and Bhadra 1990), and this approach was adopted for the identification of carcinoscorpin. The lectin was purified from freshly drawn haemolymph of C. rotundacauda (Dorai et al 1981; Abidi et al 1987) and the antisera was raised in rabbits. This was used to incubate with haemolymph containing the lectin for the immunological identification of carcinoscorpin.
2.2 Organism used for experimental infection
Natural pathogen for the marine horseshoe crabs have not been reported, but administration of Gram-negative E. coli resulted in the death of the horseshoe crab by fatal intravascular clot formation as reported long back in works with L. polyphemous. So E. coli was used as the experimental pathogen. From the Applied Microbiology Section of this Institute E. coli K-12 was collected to conduct the experimental infection by administering the live bacteria into the horseshoe crab. The bacteria was grown overnight in the nutrient broth, harvested, washed thrice in pyrogen free sterile 0.85% NaCl solution and finally suspended in the same solution to a cell density 1010 CFU/ml and stored at 4oC till use.
2.3 Inoculation of C. rotundacauda with live E. coli
The stock suspension of E. coli was washed thrice and diluted with pyrogen free 3% NaCl solution on the day of use to adjust the required cell density, ranging from 103 to 1010 live bacteria/ml. Freshly prepared 1 ml suspension of the bacteria with known count was injected into the cardiac chamber of the live crab slowly taking 23 min to complete the administration. As the first peak of lectin induction appeared at 2 h after the administration of Kdo, this was the time point at which the bacteria were introduced into the live crabs to assess the capacity of the host to defend itself. The control group having no induced lectin was also treated similarly with identical dose of live bacteria while the third control received in parallel pyrogen free sterile 3% saline. The survival of the crabs were then recorded for all the groups as stated before (Basu Sarbadhikari and Bhadra 1990).
2.4 In vitro killing of E. coli
Live E. coli (stock suspension) was diluted as stated above with sterile lectin binding buffer (50 mM Tris HCl, pH 7·8, 10 mM CaCl2) made in pyrogen free 3% NaCl solution. For the in vitro killing, each ml of the bacterial suspension containing 107 live bacteria was incubated with 400 m g pure carcinoscorpin. A parallel set of the bacterial suspension, with no added lectin, was used as the control. At the end of a 45 min incubation at room temperature the bacterial samples were centrifuged (at 10,000 g for 10 min at 4°C) to get the bacterial pellet. Then four samples in duplicate were incubated with the lectin binding buffer, the same buffer having 400 m g/ml pure carcinoscorpin, and with or without the suspension of freshly prepared amoebocytes of C. rotundacauda, at a ratio of bacteria : amoebocytes, 16 : 1, at room temperature. To collect the amoebocytes freshly drawn haemolymph was diluted in a pyrogen free saline EDTA citrate buffer (50 mM TrisHCl, pH 7·8, 10 mM EDTA, 30 mM Na-citrate and 3% NaCl). The cells were then collected by centrifugation (150 g, 1 min) at room temperature. They were washed twice with pyrogen free 3% NaCl solution and suspended in the same saline made into sterile Krebs Ringer glucose buffer (Elliot 1955). The amoebocyte population was adjusted by direct microscopic count so that the desired ratio was obtained during the incubation with E. coli. Aliquots of 100 m l were withdrawn from all the incubations at the specific time and diluted for plating on nutrient agar and one drop for microscopic examination. Giemsa staining was carried out to see the integrity of amoebocytes.
2.5 In vitro phagocytosis of E. coli
Horseshoe crab amoebocytes, interacting with trace amounts of Gram-negative bacterial endotoxin or lipopolysaccharide undergo instantaneous disintegration with the concomitant release of intracellular clotting system (Levin 1976). Attempts to illustrate in vitro phagocytosis of Limilus amoebocytes were reported unsuccessful (Armstrong and Levin 1979).
The amoebocytes of C. rotundacauda, like those of Limulus were shown to be equally sensitive to endo-toxin mediated disintegration (Mahalanobis et al 1979). However, under pyrogen free conditions, it was possible to fix amoebocytes on microscopic slides very quickly by forced air drift from a hair drier. These amoebocytes were found to be stable at room temperature for more than 30 min before suffering from disintegration. Attempts were made to use these amoebocytes for the demonstration of phagocytosis of E. coli (in terms of the number of bacteria/or its aggregate taken up).
In a typical experiment, a drop (50 m l) of freshly drawn haemolymph was mixed with 50 m l pyrogen free NaCl solution on a pyrogen free microscope slide. The slide was then tilted up and down to spread the drop. A thin film thus formed on the slide was held 45 cm away from a hair drier. The air blow hit the thin liquid film on the slide. Within 1 to 1·5 min the film was semidry. Microscopic examination confirmed the integrity and viability of the amoebocytes based on trypan blue exclusion test safely over 30 min . The bacterial suspension treated with or without carcinoscorpin as stated before was mixed with the slide adhered amoebocytes present in the haemolymph film. The density of the bacterial suspension was adjusted so that the ratio of the bacteria to amoebocytes was in the range of 1 : 70 to 1 : 80. The slides were then placed in a humid chamber at 37°C. At a specified time two to three slides were taken out, washed with chilled pyrogen free NaCl solution (3% NaCl and 10 mM CaCl2) and examined microscopically. Amoebocytes stained with Giemsa were used for viewing under microscope. The number of bacterial aggregate attached to or ingested by an amoebocyte was calculated and the results were expressed as the average of fifty amoebocytes counted.
3.1 Role of induced carcinoscorpin
on the survival
of the horseshoe crab (C. rotundacauda) after
challenge with live E. coli
In the experimental group, the horseshoe crabs were inoculated with live E. coli at 2 h, the time point of the appearance of a minor (2-fold) peak of induced lectin and also at 4 to 6 h when the induced level of carcinoscorpin rose to 816 times of the normal circulatory titer. One control group was treated similarly with live E. coli and another with 3% sterile pyrogen free saline. Since the time of administration of the bacteria the survival period of the crabs was recorded up to 96 h as shown in figure 1. The horseshoe crabs being administered with live E. coli up to a dose of 106 live bacteria per animal survived throughout the period of observation in all groups. While the experimental group having 816-fold induced circulatory carcinoscorpin survived throughout the same period having been administered with an average infective burden of 107 live E. coli per crab. Thus those crabs which had 816-fold induced circulatory lectin, were found capable of combating a 10-fold higher infective bacterial load compared to saline injected control. A capacity to tolerate 9 million more of live E. coli was acquired by the groups having induced lectin. When the dose of administration exceeded 107 live E. coli per crab, the control groups having no induced lectin died much earlier than the one with 816-fold induced lectin. For example the one having 816-fold induced carcinoscorpin showed a period of survival on average nearly three times longer than what was observed for the control having no induced lectin after receiving 1010 live E. coli, the highest infective dose used here (figure 1). Therefore, the elevated level of the circulatory carcinoscorpin in the Kdo administered group seemed to correlate well with increased protective immunity or ability of C. rotundacauda against an experimental infection
3.2 Influence of carcinoscorpin on
in vitro killing
of the pathogen, E. coli
Opsonization of live E. coli with carcinoscorpin by incubating in a lectin solution (1 mg/ml of 50 mM Tris HCl, pH 7·8, 10 mM CaCl2, 150 mM NaCl) showed some loss of viability compared to unaffected viability for the samples treated in parallel with binding buffer only. The bacterial killing increased with the progress of incubation in the presence of freshly prepared Carcinoscorpius amoebocytes. Carcinoscorpin opsonized bacteria showed approximately 33% more killing, compared to the non-opsonized samples (figure 2). The amoebocyte-bacteria mixed suspensions however turned viscous gradually irrespective of the prior treatment with carcinoscorpin. It took 45 min for this change to occur possibly due to degranulation of the internal content of the amoebocytes and triggering of the clotting cascade. Microscopic examination of the incubation mixture revealed the loss of the cellular integrity for most of the amoebocytes within 5 min of incubation, but bacterial killing continued during the entire 30 min, the period of incubation fixed for the observation (figure 2).
3.3 Role of carcinoscorpin on the phagocytosis of E. coli
Live bacteria, after incubation with or without lectin carcinoscorpin, were subjected to the phagocytosis by circulatory amoebocytes adhered onto microscopic slides (figure 3). The bacterial suspension irrespective of its opsonization with carcinoscorpin, when mixed with the slide adhered amoebocytes in the thin haemolymph film, was exposed to lectin present in the semi dried film. In situ bacterial aggregate formation resulted in discrete forms on all the slides. But there was a clear difference in the number of bacteria or aggregate ingested by the amoebocytes for the lectin treated and untreated bacterial preparation. On an average an amoebocyte showed nearly 1·5 times higher take up of the bacterial aggregate for lectin opsonized samples.
In the earlier work carcinoscorpin induction by administering haptenic Kdo and b -glycerophosphate into the live crabs (Basu Sarbadhikary and Bhadra 1990) and inhibition of Gram-negative E. coli agglutination by Kdo (Dorai et al 1982) were suggestive for the physiological significance of the lectin in relation to the defence of the host. As the haptenic challenge mediated induction of carcinoscorpin is somewhat synonymous with antibody production by antigen challenge in higher vertebrate the lectin dependent bacterial agglutination is also comparable to the vertebrate antibody mediated cell agglutination.
Indian horseshoe crab, C. rotundacauda, an invertebrate belonging to the family of arthropod, is assumed to rely on innate immunity for the host defence (Söderhäll and Cerenius 1998). It was also proposed that it would occur on the basis of some sort of recognition of the invaders for the production of appropriate immune factor.
In this study circulatory lectin level dependent host defence of the horseshoe crab C. rotundacauda has been demonstrated, where the production of the lectin was modulated by Kdo, the cell wall component of the experimental pathogen. As Kdo is a constituent of E. coli cell surface LPS (Meyer et al 1989) so clear recognition of the defined molecular component of the invader has been established here for the production of the immune factor. In the invertebrate system, a number of LPS binding proteins have been implicated in the defense of the host. For example Periplaneta lectin and LPS binding protein (LBP) which are functionally opsonins (Natori and Kubo 1996), insect blood protein hemolin as phagocytosis stimulator (Mendoza and Faye 1996), silk worm Bombyx mori Gram-negative bacteria binding protein (GNBP) (Lee et al 1996), lipophorin, blood LPS removing protein (Kato et al 1994), and horseshoe crab factor C, a LPS induced clotting agent (Kawabata et al 1996) have been characterized at the molecular level and found to differ greatly in their chemical composition. The defined component of LPS involved in the binding interaction of these proteins is unknown and the regulation of their production has remained far from our understanding.
In C. rotundacauda not only the molecular basis of the induction of the circulatory lectin has been demonstrated but also a quantitative relation between the circulatory level of the lectin and the extent of protection has emerged. About 10-fold (816-folds) induction of the lectin enhanced the defense of the host in such a way that it was able to resist the infection with 10-fold higher pathogen. The natural circulatory level of the lectin provided the host a basal protective capacity for combating 106 live E. coli and such a level of infection is assumed to be uncommon. Its function as an opsonin has been well clarified when the bacterial killing was noticed after incubation with the lectin beside its agglutinating activity which was already known. From B. mori an inducible Gram-negative bacteria binding has been reported as a component of host defence (Lee et al 1996) and another LPS-binding protein involved in the bacterial clearance from the haemolymph has recently been reported (Koizumi et al 1997). In vitro killing of lectin opsonized bacteria was significantly increased in association with isolated crab amoebocytes. The role of carcinoscorpin on the higher phagocytic uptake was clearly demonstrated though such a demonstration was unsuccessful for Limulus (Armstrong and Levin 1979). Quick fixing of haemolymph amoebocytes by forced air drift was made accidentally and found very rewarding for having the amoebocytes stable for 30 min and capable of phagocytic uptake. Lectin opsonized bacteria showed much higher uptake by the phagocytic amoebocytes. Thus the opsonizing role of the lectin was further strengthened and acted like a vertebrate humoral factor. The receptor for lectin on the amoebocytes remained unknown but an activity modulated state of the amoebocytes has been evidenced in this case. Animal coagulating system under ex vivo condition is highly sensitive to external stimuli since amoebocytes were easily disintegrated with the induction of coagulation in the bacterial incubation system. On the basis of this finding it can be proposed that in vivo state of the amoebocytes might have the fate like the one attained in in vitro phagocytosis here and the lectin might act functionally as an opsonin.
Carcinoscorpin, the circulatory lectin of C. rotundacauda is related closely to limulin of Limulus polyphemus, an invertebrate protein with high degree of functional homology with C-reactive protein of vertebrates (Robey and Liu 1981). But carcinoscorpin unrelated to C-reactive protein activity is produced by injury to C-reactive protein and its site of synthesis is hepatopancrease (Basu Sarbadhikari et al 1995) while that of C-reactive protein is vertebrate liver. It is functionally a primitive recognition molecules like the members of the pentraxin family (Marchalonis and Schluter 1989). The leukocyte surface protein L-selectin expressed by inflammation has lectin like carbohydrate recognition domain or CRD (Feizi 1993), and the carbohydrate that is recognized by the L-selectin CRD is identical with the carbohydrate recognized by carcinoscorpin (Abidi et al 1987).
Carcinoscorpin of C. rotundacauda appeared to have multiple functional role relevant to the immune response as a humoral factor. It is plausible to think that in the primitive world when evolutionary differentiation was not distinct some functional protein domain might have evolved and segregated later retaining it even in the higher vertebrate.
We thankfully acknowledge the financial support of
the University Grant Commission (DD) and the Indian Council of Medical Research (SBS), New Delhi.
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MS received 5 March 1998; accepted 27 July 1999
Corresponding editor: Samir Bhattacharya
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