Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

JOURNAL

OF

INVERTEBRATE

PATHOLOGY

31, 164-170 (1978)

Opsonizing Effect of Serum and Albumin Gland Extracts on the Elimination of Human Erythrocytes from the Circulation of Helix poma tia LOTHARRENWRANTZ'ANDWOLFGANG Institute

for

MOHR

Pathobiology, Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania and Zoologisches Institut und Zoologisches Museum der Universifiit Hamburg. Martin-Luther-King-Platz 3. 2 Hamburg 13. West Germany

18015,

Received March 24, 1977 The removal of human erythrocytes of the A, and B types from the circulation of the gastropod pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A, and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A, erythrocytes injected 12-19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A, erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A, erythrocytes in the agglutinating extract of the sponge Aaptos papillata. KEY WORDS: Helix pomatia; Cepaea (Helix) nemoralis: Aaptos papillata; human erythrocytes; serum opsonin; albumin glands; kinetics; serum: opsonizing effect. Helix

INTRODUCTION

The importance of phagocytosis as an immune mechanism in invertebrates is well established; however, the process by which phagocytes are able to recognize foreign particles remains essentially undetermined. It is known that in vertebrates two mechanisms are involved in the attachment of nonself materials to the surface of phagocytes: Foreign particles may either interact directly with phagocytic cells, or opsonins may mediate their recognition (Hirsch, 1975). Opsonization in vertebrates is primarily a function of the third component of complement (C,) or IgG and IgM antibodies (Berken and Benacerraf, 1966; Stossel, 1972; Frank et al., 1975). After the invading microorganisms become coated with these molecules, the opsonin-particle complex becomes coupled to the surface of phagocytes. The receptors for C, and IgG on 1 Visiting Research Scientist from the Zoologisches Institut und Zoologisches Museum der Universitlt Hamburg, West Germany, sponsored by the Deutsche Forschungsgemeinschaft. 0022-201 l/78/0312-0164$01.00/O Copyright All rights

0 1978 by Academic Press. Inc. of reproduction in any form reserved.

164

mammalian macrophages and polymorphonuclear neutrophils have been identified (Berken and Benacerraf, 1966; LoBuglio et al., 1967; Huber et al., 1968; Scribner and Fahrney, 1976). Although various in vitro observations on the opsonizing activity of invertebrate serum have been published (Tripp and Kent, 1967; Stuart, 1968; Prowse and Tait, 1969; McKay and Jenkin, 1970; Pauley et al., 1971), it is still unclear if opsonins are of general importance for the clearance of foreign particles from the circulation of invertebrates. Also, the chemical nature of invertebrate opsonins has not yet been characterized; however, there is some evidence that naturally occurring agglutinins are involved in the recognition process (McKay and Jenkin, 1970; Anderson and Good, 1975). In order to gain more information on the molecular basis of recognition as related to immunity in invertebrates, experiments were performed to investigate the kinetics of the clearance of human erythrocytes in Helix pomatia, and to ascertain the in-

OPSONINS IN HELIX

fluence of humoral factors on the elimination of human erythrocytes of the A, and B types from the circulation. In addition, studies were carried out to ascertain whether the serum opsonin level has a limiting effect on the rate of clearance of erythrocytes comprising a second challenge. Finally, the clearance rates of human A, erythrocytes that had been incubated in different anti-A agglutinin-containing solutions were ascertained. The agglutinating extracts employed were prepared from the albumin glands ofH. pomatia (Prokop et al., 1965; Hammarstrom and Kabat, 1969,197l) and Cepaea (Helix) nemoralis (Schnitzler and Kilias, 1970; Krtipe and Pieper, 1966) and from the sponge Aaptos papiffara (Bretting, 1973; Bretting and Renwrantz, 1974). MATERIALS

AND METHODS

Snails. The specimens of Helix pomatia used were purchased from Robert Stein Co., Lauingen, West Germany. The snails were either stored in the hibernation state at 6°C or were kept under natural conditions outside the institute. Hibernating snails were reactivated at room temperature 2 days prior to injections. They were maintained in cages the bottoms of which were covered with moist paper towels. Animals that lived under natural conditions were placed in similar cages l-2 hr prior to injections. All of the snails were fed ad libitum on lettuce. Erythrocytes. Human A, and B erythrocytes were washed four times in a 20-fold excess of physiological saline. We prepared cell suspensions at known concentrations by using a Thoma hemocytometer. Incubation media. The serum of H. pomatia was collected in the following manner: The shells of hibernating snails were thoroughly cleaned and a piece was removed in order to expose the lung. A lateral pulmonary sinus was then punctured and the snail was placed over a 25-ml beaker. The pooled hemolymph was centrifuged at 1OOOg for 10 min to remove the

POMATIA

165

cells. The serum was decanted and used the same day in our opsonization experiments. Albumin glands of H. pomatia and C. nemoralis were extracted as previously described (Renwrantz et al., 1975). The Aaptos papillatu extracts were prepared according to Bretting and Renwrantz (1974). Incubation of the erythrocytes. To sensitize the erythrocytes, 1 vol of packed cells was incubated for 1 hr at room temperature with 20 vol of each of the following preparations: (1) H. pomatia serum, (2) a 1:600 dilution of the H. pomatia albumin gland extract (the initial titer was 1:128), (3) a 1:1200 dilution of the C. nemoralis albumin gland extract (the initial titer was 1:256), and (4) a I:4000 dilution of the Aaptos extract (the initial titer was 1: 1024). The agglutinin titers were determined against human A1 erythrocytes by employing the Takatsy microtiter system. Injections. An 0.2-ml sample of standardized erythrocyte suspensions (see Results) was injected by use of a sterile syringe into the hemocoel in the upper subepithelial region of the headfoot. Measurement of hemolymph clearance.

The rate of clearance of injected erythrocytes was measured by examining up to 10 hemolymph samples within a maximum period of 3.5 hr. In order to collect hemolymph samples, the columellar sinus was punctured with a needle and the hemolymph which immediately oozed out was collected with a calibrated glass pipet (average, 50 ~1). After the collection of each sample, a small amount of tissue glue (Histoacryl Blau, B. Braun Melsungen AG, West Germany) was placed on the punctured sinus, and the immediate polymerization of the glue prevented continuous bleeding. Clearance curve and phagocytic

index.

The clearance of injected erythrocytes is a first-order kinetics reaction which follows an exponential function over time and can be expressed by the equation: y = A-en”. By using the determined erythrocyte concentrations of the single hemolymph

166

RENWRANTZ

samples (Yi) and the time after the injection at which these samples were taken (ti), one can compute the values of the coefficient A and the exponent B, thus obtaining the calculated exponential function which best fits the plotted points. The computation of A and B was performed with the help of a Wang computer using the Wang 2000 Series Program-Exponential Regression (Roscoe, 1969). For each experimental series, a mean clearance curve was calculated by using the means A and B from the A and B values of n individual clearance curves. According to the methods of Biozzi et al. (1953) and Benacerraf et al. (1959), the mean phagocytic index (k) was calculated from the slope of the logarithmic form of a mean clearance curve by employing the equation K = (log c, - log C,)/(fB - tl), in which C, and CZ are the erythrocyte numbers of the hemolymph at the times tl and t2. The standard deviation of the mean phagocytic index R was determined by substituting the mean A and B + SDS or B - SDa into the exponential function. RESULTS

Kinetics

of Hemolymph

Clearance

The kinetics of erythrocyte clearance from the hemolymph of H. pomatia follows

FIG. 1. (a) Clearance exponent B = -0.01993.

of B erythrocytes;

(b) logarithmic

AND

MOHR

the same pattern as does the clearance of particles from the circulation of vertebrates (Biozzi et al., 1953; Benacerraf et al., 1959). In order to demonstrate the characteristic pattern of the removal of injected B erythrocytes, in Figure la are depicted the results obtained with one snail. After the injection of 0.2 ml of a B-cell suspension containing 1.6. lo9 cells, we withdrew 10 hemolymph samples over a period of 186 min. The rate of clearance was K = 0.0087. It was determined from the logarithmic form of the clearance curve (Fig. lb). Clearance of Primary Doses of Nonsensitized and Serum-Incubated A, and B Erythrocytes A comparison of the clearance rates of nonsensitized and serum-incubated A, erythrocytes indicates a faster elimination of the incubated cells (Table 1). By employing the Student’s t test, it was determined that the phagocytic indexes were significantly different (P < 0.05). The conclusion that the H. pomatia serum contains serum factors which have opsonic ability was confirmed by the finding that preincubation in serum also causes a strong increase in the rate of removal of B erythrocytes. The difference between the phagocytic indexes of nonsensitized and serum-pretreated B cells (Table 1) is highly significant (P < 0.02).

form

of the clearance

curve.

Coefficient

A

= 48.7;

OPSONINS IN HELIX TABLE

1

TABLE

CLEARANCE RATES OF PRIMARY DOSES OF NONSENSITIZED AND SERUMINCUBATED HUMAN ERYTHROCYTES

Injected erythrocytes Type A, Nonsensitized A, Serumincubated B Nonsensitized B Serumincubated

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POMATZA

3

CLEARANCE RATES OF SECONDARY DOSES OF ERYTHROCYTES WHICH WERE INCUBATED IN DILUTED AGGLUTININS PRIOR TO INJECTION

Injected erythrocytes

Concennation

Number of snails

Phagocytic index (k)

1.1,109

14

0.0164 _t 0.0029

1.1.109

10

0.0187 2 0.0023

1.6. lo9

13

0.0085 t 0.0012

1.6.109

7

0.0101 _f 0.0016

Type A, Incubated m HP,,,” A, Incubated in CN,,,* A, Incubated in AF

Concentration

Number of snails

Phagocytic index (k)

l.l.lOY

9

0.0346 lr 0.0067

1.1.109

5

0.0249 f 0.0013

1.1.109

5

0.0082 +- 0.0004

u HP,,,: Albumin gland extract of Helix Albumin gland extract of Cepuea c AP: Extract of Aaptos papillata. * CN,,,:

Clearance of second doses of nonsensitized and serum-incubated A, Erythrocytes

Twelve to nineteen hours after a primary injection of 1.1. log A, erythrocytes, the snails received a second dose of the same quantity of either nonsensitized or serumincubated A, cells. As indicated by the phagocytic index (Table 2), nonsensitized secondary doses were cleared very slowly from the snail’s circulation. Serum-incubated erythrocytes comprising the second doses (R = 0.0149), however, were removed at a rate (P > 0.2) similar to the primary doses of nonsensitized A, erythrocytes (k = 0.0164). TABLE

2

CLEARANCE RATES OF SECONDARY DOSES OF NONSENSITIZED AND SERUM-INCUBATED A, ERYTHROCYTES

A, Nonsensitized A, Serumincubated

pomuria. nemoralis.

Influence of Agglutinins on Clearance of Second Doses of A, Erythrocytes

Twelve to nineteen hours after a primary administration of 1.1. lo9 A, erythrocytes, the snails were injected with the same quantity of A, cells which had been preincubated in albumin gland extracts of N. pomatia or C. nemoralis or in an extract of A. papillata. The three solutions were used in subagglutinating concentrations. The results of these tests are summarized in Table 3. It is apparent that only the albumin gland extracts showed a strong opsonizing effect. The clearance rates of A, erythrocytes which were incubated in Aaptos extract (k = 0.0082) were not statistically different (P > 0.5) from the clearance rates of second doses of nonsensitized A, cells (R = 0.0078). DISCUSSION

Injected erythrocytes Type

A,

Concentration

Number of snails

Phagocytic index (K)

1.o. 109

8

0.0078 -+ 0.0013

l.l.lOY

8

0.0149 + 0.0019

The removal of carbon particles and bacteria from the circulation of H. pomatia has been investigated by Reade (1968) and Bayne and Kime (1970), respectively. The clearance pattern described by these authors and the results of the studies presented herein indicate that the rate of

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RENWRANTZ

elimination of nonself particulate material in the snail follows an exponential curve as has been found in a variety of vertebrates (Biozzi et al., 1953; Rogers and Melly, 1957; Benacerraf et al., 1959; Jenkin and Rowley, 1961; Nelstrop et al., 1968) and in the crayfish Purachaeraps bicarinatzts (Reade, 1968; Tyson and Jenkin, 1973). Furthermore, our studies have shown that the elimination of certain foreign particles by the phagocytic system of H. pomatia is strongly influenced by hemolymph components of the snail. Nonsensitized human A, (R = 0.0164) and B erythrocytes (/? = 0.0085) were cleared significantly slower than were A, (R = 0.0187) and B cells (I? = 0.0101) which had been incubated in Helix serum prior to injection. It is not yet known whether one or more components are involved in the opsonization of A, and B erythrocytes. The elimination of erythrocytes comprising the second challenge from the circulation of H. pomatia is similar to the secondary clearance pattern observed in different vertebrates. A depressed clearance rate was reported in mice when second doses of particles were given 30- 120 min after the primary injection (Jenkin and Rowley, 1961). It was shown that this reduced activity was caused by a depletion of serum opsonins. However, when primary and secondary injections were made more than 48 hr apart, an increase in the secondary clearance rate was observed, apparently triggered by an increase in the level of opsonizing serum antibodies (Biozzi et al., 1953; Benacerraf et al., 1959; Jerne, 1960: Nelstrop et al., 1968). In part, results comparable to those just cited were observed in the crayfish Parachaeraps bicarinatus by Tyson and Jenkin (1973). They found that primary doses of particles adsorbed circulating serum opsonins so that secondary doses were removed more slowly from the crayfish blood. Likewise, in H. pomatia, the elimination of A, erythrocytes comprising the second challenge (R = 0.0078) was significantly

AND

MOHR

slower than the clearance rate of primary doses (k = 0.0164). However, when the second dose was pretreated with serum the elimination rate returned to normal (k = 0.0149). From these results, it is concluded that opsonins play a key role in the recognition of certain foreign particles by the phagocytic system of H. pomatia. The strong opsonin dependence of the clearance of A, erythrocytes becomes more understandable when one considers that a direct attachment of human red cells to Helix phagocytes essentially does not occur (Renwrantz and Cheng, 1977b). Our results also show that one limiting factor in the uptake of high quantities of foreign particles may be the amount of opsonizing molecules in the serum. However, it should be stated that opsonins can only enable or enhance phagocytosis as long as the phagocytic system is not saturated. Studies by Munthe-Kaas (1976) of phagocytosis in rat Kupffer cells have indicated that the final limiting factor in the uptake of foreign particles is the total volume of the phagocytic cells. Although the opsonizing effect of H. pomatia serum has been demonstrated, the chemical nature and the origin of serum opsonin(s) still remain undetermined. However, there is good evidence that naturally occurring hemagglutinins may serve as opsonins (McKay and Jenkin, 1970; Anderson and Good, 1975). While the serum of H. por?zatin contains a very weak agglutinin, which can only be detected by enzymetreated indicator cells (Reifenberg and Uhlenbruck. 1971; Renwrantz, 1974). a strong anti-A agglutinin has been found in the albumin gland of this snail (Prokop et al., 1965: Hammarstrom and Kabat, 1969, 1971). Since H. ponzatia has an open circulatory system, it cannot be excluded that the albumin gland releases molecules into the surrounding body fluid. Consequently, we investigated albumin gland extracts of H. pomatia with respect to their opsonizing activity. As the results show, the incubation of erythrocytes com-

OPSONINS IN HELIX

prising the second challenge in the extract (subagglutinating concentration) even caused a stronger increase in the clearance rate (k = 0.0346) than presensitizing these erythrocytes with serum (k = 0.0149). Two other A, erythrocyte-agglutinating solutions showed different effects on the phagocytic activity of the snail. While the albumin gland extract of C. tlemorulis was strongly opsonic (I? = 0.0249), the agglutinin of A. papillata did not enhance the clearance of secondary doses (Z? = 0.0082). Results similar to those have been obtained by McKay and Jenkin (1970) in the crayfish, P. bicarinatus. Only the crayfish agglutinin and the agglutinating serum of a crab were opsonic. Sera from species of other phyla did not enhance the phagocytosis of sheep erythrocytes by this crayfish. As it is known that opsonins function as connecting bridges between particles and the surface of phagocytes (LoBuglio et al., 1967; Huber et al., 1968), the nonopsonizing effect of some agglutinins can possibly be explained by a lack of phagocyte receptors specific for these agglutinating molecules (Renwrantz and Cheng, 1977a). The observation that the plant agglutinin Con A may facilitate phagocytosis of mouse erythrocytes by vertebrate macrophages (Goldman and Cooper, 1975) does not support the conclusion that the “recognition factors may be specific for each phyla” (McKay and Jenkin, 1970). This investigation does not contribute to the question of the nature of the phagocytic system of H. pomatia that is responsible for the clearance of small nonself particles. Studies are in progress to clarify if circulatory hemocytes are eliminating injected erythrocytes or whether, as suggested by Bayne (1974), other mechanisms, i.e., fixed phagocytes, phagocytic cells of the digestive gland, are clearing the hemolymph of H. pomatia. ACKNOWLEDGMENTS We wish to thank Dr. T. C. Cheng and Dr. T. P. Yoshino for correcting our English in the manuscript.

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POMATIA

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