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Excretory-secretory (E-S) products contained in supernatants from in vitro cultured Schistosoma mansoni primary sporocysts were assayed for their effects on the in vitro motility of Biomphalaria glabrata hemocytes. Both whole (unfractionated) and fractionated E-S products were tested in modified Boyden chemotaxis chambers. E-S product fractionation was accomplished using both membrane ultrafiltration (MF) and high-pressure liquid chromatography (HPLC). Transformation (Tr) products, but not those products released by 8-day sporocysts, significantly inhibited the random motility of hemocytes from an S. mansoni susceptible strain (M-Line) of B. glabrata. This activity was found in both high and low MF fractions of Tr but not in an intermediate MF fraction. In an effort to isolate the active component(s) of the high MF fraction, HPLC was used to separate components based on size exclusion. Although each of four HPLC fractions displayed some inhibitory activity, the greatest consistent activity was found in fraction 3, which was composed, predominantly, of a 108-kDa protein. In contrast to the response of M-Line cells to Tr E-S products, the motility of hemocytes from an S. mansoni-resistant strain (10-R2-OK) of B. glabrata was not significantly reduced from controls. The high MF fraction, however, elicited a slight positive chemokinetic response, while the low MF fraction reduced 10-R2-OK hemocyte motility slightly but not significantly. While three HPLC fractions significantly reduced 10-R2-OK hemocyte motility, this effect was significantly less than that produced by the same HPLC fractions on M-Line hemocyte motility. These data suggest that S. mansoni sporocyst Tr E-S products differentially affect the random motility of M-Line and 10-R2-OK snail hemocytes. Although the significance of this differential effect on the in vivo defenses of B. glabrata is not known, it could be important in the host-parasite interaction which leads to either resistance or susceptibility.

Five monoclonal antibodies (mABs) against surface antigens on circulating, glass-adherent hemocytes of the snail, Biomphalaria glabrata, were produced by somatic cell hybridization methods. Two mABs (IID2.6-Bg and IID4.8-Bg) are pan-hemocytic, reacting uniformly with epitopes shared by all adherent hemocytes. Determinants recognized by these mABs also are present in soluble form and appear to be associated with a hemoglobin-depleted ultracentrifuged fraction of snail hemolymph. Hybridoma-derived mABs IIC6.8-Bg and VB10.3-Bg recognize hemocyte surface epitopes expressed by only 50-60% of the adherent cell population. These mABs also are reactive with soluble hemolymph antigens but apparently recognize determinants which are different from the IID2.6-Bg and IID4.8-Bg reactive sites. Another antigenically distinct hemocyte subpopulation is recognized by mAB IID7.1-Bg. Epitopes that are reactive with this mAB differ from the previously described determinants by their asymmetrical distribution on the surface of positive cells and the absence of soluble antigenic components in hemolymph. Furthermore, unlike the other mABs, the prevalence of hemocytes staining with IID7.1-Bg antibodies differed between two strains of B. glabrata. Results of this study clearly demonstrate that circulating B. glabrata hemocytes, consisting of a single, predominant population of adherent cells, is composed of several distinct antigenic subpopulations based on the specific binding of anti-hemocyte mAB probes. Our successful application of hybridoma techniques to the study of molluscan hemocyte surface antigens underscores further the great potential usefulness of this method in analysing the molecular basis of hemocyte reactivity.

Serum-free hemocytes from the clam, Corbicula fluminea, are cytolytic to mammalian erythrocytes (RBCs) as demonstrated in a hemoglobin release assay. The reaction is hemocyte dose- and temperature-dependent, and is mediated by the release of soluble hemolytic factors from clam cells. Initiation of hemocyte secretory activity does not appear to require contact with target RBCs. The chemical nature of the secreted lysin(s) has not been determined; however, PAGE analysis of hemocyte extracts (lysates) reveals the presence of at least five peaks of lytic activity. This activity is sensitive to heat and proteolytic enzyme treatment, and can be removed by absorption with fixed, homologous RBCs. Thus it is likely that the secreted hemocyte lysin is a heat-labile protein which exerts its hemolytic activity by binding directly to target RBC membrane constituents.

The fate of surface concanavalin A (Con A)-receptor complexes on hemocytes from two stocks of the snail, Biomphalaria glabrata (PR albino and 10-R2), was studied using both direct (Con A-FITC) and indirect (biotin-Con A, avidin-FITC) fluorescent staining methods. The results of experiments in which living hemocytes were exposed first to biotin-Con A, followed by fixation and subsequent treatment with avidin-FITC to localized surface-bound lectin, demonstrate that Con A complexes are rapidly cleared from hemocyte plasma membranes. Experiments employing Con A-FITC further indicate that clearance is accomplished through an internalization process whereby Con A-complexes are endocytosed and eventually accumulate as a single intracellular aggregate of fluorescent material in the cell body of each hemocyte. In addition, a redistribution of surface complexes into patches and caps is concomitant with receptor clearance, with a maximum frequency of patching (approx. 45%) and capping (approx. 30%) occurring within 5 and 10 min., respectively, of initial Con A exposure. PR albino and 10-R2 snail hemocytes appear indistinguishable with regards to their responsiveness to Con A-binding. The close similarity in the behavior of Con A complexes or circulating hemocytes from both snail stocks further support the conclusion that Con A-reactive determinants and their associated membrane components probably represent identical molecular populations on PR albino and 10-R2 B. glabrata blood cells.

Circulating hemocytes of the snail, Biomphalaria glabrata, synthesize and secrete a variety of polypeptides when maintained in vitro in serum-free medium containing [35S] methionine. SDS-PAGE/fluorographic analysis of supernatants from resistant snail (10-R2-OK strain) hemocyte cultures revealed the presence of numerous labeled polypeptides ranging in Mr from 220 to 14 kDa. Most of these same proteins were also produced by hemocytes of a susceptible B. glabrata strain (M-line), but the overall rate of secretory protein synthesis was reduced from that of resistant snail cells. In addition, excretory-secretory (ES) products contained in supernatants from Schistosoma mansoni miracidial transformation and 1-day primary sporocyst cultures stimulated increases in the synthesis of various polypeptides. Particularly striking was a 3-fold increase in the synthesis of a 66-kDa secretory polypeptide by hemocytes of both snail strains, and a concomitant increase in M-line hemocytes and decrease in 10-R2-OK cells of a 63-kDa polypeptide. Overall, however, the level of ES product-induced secretory protein synthesis was greater in 10-R2-OK snail hemocytes than in those of the M-line strain. Exposure of a nonhemocytic B. glabrata cell line to parasite culture supernatants had no stimulatory/inhibitory effect on labeled protein ouput, suggesting that the observed hemocyte response may be snail cell-type specific. Finally, the larval ES components responsible for modulating hemocyte protein metabolism are mainly concentrated in a heat-stable fraction composed of molecules of greater than 30 kDa. However, the loss of the ability of heated parasite products to stimulate synthesis of certain hemocyte proteins and the presence of minor stimulating activity in a low molecular weight fraction (less than 10 kDa) implies the possible existence of multiple larval components affecting formation of specific hemocyte secretory polypeptides. It is concluded that snail hemocytes are capable of in vitro synthesis and secretion of a variety of methionine-containing polypeptides, and that ES products of early larval schistosomes can modulate (i.e., stimulate or inhibit) this metabolic process. A differential response of susceptible vs. resistant hemocytes to larval products suggests that the degree to which these cells can be metabolically activated may determine their cytotoxic effectiveness.