Fc receptor-independent phagocytosis by mouse macrophages of erythrocytes sensitized with a lectin from a marine invertebrate.

A lactose-specific lectin present in the haemolymph of the colonial ascidian Botrylloides leachii was tested for its ability to act as an opsonin for the phagocytosis of erythrocytes by mouse macrophages. Sensitization of sheep or mouse erythrocytes with one agglutinating dose of the invertebrate agglutinin enhanced significantly the clearance of these cells from the circulation of mice, as did treatment with specific antibody. By measuring the adhesion and phagocytosis of 51Cr-labelled erythrocytes by monolayers of macrophages in vitro, it was shown that the B. leachii agglutinin was highly opsonic, promoting the ingestion of erythrocytes by the macrophages with an initial efficiency similar to that of specific antibody. The adherence and ingestion of antibody-coated erythrocytes was severely inhibited by blocking Fc receptors on the macrophages with soluble immune complexes, whereas the binding and ingestion of lectin-coated erythrocytes was unaffected by this treatment. The results demonstrate that invertebrate lectins can act as efficient opsonins for the uptake of particles by vertebrate phagocytes and that it is not necessary for particles to bind to Fc receptors for phagocytosis to be induced.

Suppression of early immunity to Nematospiroides dubius in mice by selective depletion of neutrophils with monoclonal antibody.

The role of neutrophils and eosinophils in the acquired resistance of mice to infection with the helminth Nematospiroides dubius has been investigated in vivo by testing infected mice for their resistance to a challenge dose of larvae following treatment with monoclonal rat antibodies (MAbs) specific for mouse granulocytes. Treatment of normal and infected mice with NIMP-R10 MAb reduced the number of neutrophils in the peripheral blood and peritoneal cavity during the ensuing 2-5 days without affecting macrophage or eosinophil levels. The effect of this MAb on immunity depended on the immune status of the mice tested. The partial resistance of mice receiving a primary 'immunising' infection, followed by passive transfer of immune serum and challenge at 4 days, was completely suppressed by NIMP-R10 MAb. The acquired resistance of mice challenged 10 days after being given a single 'immunising' infection was halved by NIMP-R10 treatment, while that of 'twice-immunised' mice (infected on days 0 and 14, challenged on day 24) was unaffected. Treatment of twice-infected mice with the eosinophil-specific MAb NIMP-R6 reduced the number of eosinophils in the blood and peritoneal cavity by 40-60%, but caused no significant loss of immunity. The data indicate that after infection of mice with N. dubius, neutrophils play a predominant role in early resistance to reinfection, but they become progressively less essential as activated macrophages and (following a second infection) eosinophils become prevalent. The lack of effect of NIMP-R6 MAb treatment on the immunity of twice-infected mice may have been due to an insufficient reduction in eosinophil numbers; however, it seems likely that the neutrophils and macrophages present in these mice would have provided adequate protection even in the absence of eosinophils.

Metabolic activity of Trypanosoma lewisi cultured in vitro in the presence of normal or ablastinic rat serum.

Trypanosoma lewisi, in cultures supplemented with normal rat serum, synthesized the majority of new RNA during the first 13 hr of the culture, whereas DNA synthesis occurred from the 8th hr onwards and amino acids were incorporated into macromolecules uniformly throughout the 24 hr culture period. Thymidine was taken up by the parasite only between the 7th and 14th hr of culture, unlike uracil and amino acids which were taken up as required. Ablastin, a trypanostatic factor in the serum of infected rats, maximally inhibited DNA synthesis if it was added to the cultures before the 5th hr, partially inhibited synthesis if added between the 5th and 11th hr, but if added after this had no inhibitory effect. Ablastin only partially inhibited RNA synthesis and was without effect on amino acid utilisation.

Activation of complement by tetrathyridia of Mesocestoides corti: enhancement by antibodies from infected mice and lack of effect on parasite viability.

Tetrathyridia of the cestode Mesocestoides corti were isolated from the peritoneal cavity of infected mice. The parasites activated guinea pig and mouse complement (C) in vitro by both the classical and alternative pathways as shown by quantitative C fixation and crossed immunoelectrophoresis. The ability of tetrathyridia to activate mouse C was enhanced by preincubating the parasites in serum obtained from mice infected with M. corti. Antibodies of the IgG1 class, an immunoglobulin found in profoundly increased amounts in mice infected with M. corti, as well as IgM and IgG2 antibodies, bound to cultured tetrathyridia and facilitated deposition of the third component of C (C3) from dilute mouse serum, presumably via classical pathway activation. The results demonstrate that mouse IgG1 antibodies do not prevent the activation of C by the tetrathyridia or by C-fixing antibodies of other classes which become attached to the tetrathyridia. The activation of C in vitro by tetrathyridia did not affect their ability to grow in mice, even though C3-derived polypeptides could be detected by immunofluorescence on the surface of the parasites.

Infection of mice with Nematospiroides dubius: demonstration of neutrophil-mediated immunity in vivo in the presence of antibodies.

The ability of murine neutrophils to confer resistance to mice against infection by the helminth parasite Nematospiroides dubius has been investigated. Mice whose neutrophils had been 'altered' by an immunizing infection with third-stage larvae (L3) of N. dubius exhibited resistance to a challenge dose of L3 given 4 days after the immunizing infection, provided greater than 0.1 ml of immune mouse serum was passively transferred within 0-24 hr of challenge. Normal mouse serum was ineffective, as was immune serum given at the time of challenge to naive (unimmunized) mice. Neutrophils purified from the blood of mice infected 4 days previously were able to reduce the infectivity of exsheathed L3 when incubated with the latter in vitro in the presence of fresh immune serum. In contrast, peritoneal exudate cells collected at the same time were inactive in this test, indicating that activated macrophages capable of damaging L3 were not yet present and that the immunity of 4-day infected mice given immune serum was probably attributable to the presence of 'altered' neutrophils. The capacity of neutrophils to confer resistance to infection in vivo was unambiguously demonstrated by the passive transfer of 98 +/- 4% pure neutrophils, isolated from the blood of 4-day infected (C57Bl X BALB/c)F1 mice, to uninfected F1 mice. Only those mice which received both neutrophils and immune serum exhibited resistance to a challenge infection. In contrast, mice injected with an eosinophil-enriched cell preparation and immune serum were not resistant. These results indicate for the first time that neutrophils have the capacity to damage nematode parasites in vivo and that they are active against N. dubius in mice infected with this parasite.

Reduced infectivity of Nematospiroides dubius larvae after incubation in vitro with neutrophils or eosinophils from infected mice and a lack of effect by neutrophils from normal mice.

Neutrophils and eosinophils, isolated from the blood of mice infected with Nematospiroides dubius, were tested for their capacity to damage exsheathed third stage N. dubius larvae in vitro. In the presence of fresh serum from infected mice, both types of granulocyte caused a significant reduction in larval infectivity (up to 40-50%) whereas lymphocytes/monocytes prepared from the same blood samples were inactive. Neutrophils were at least as active as eosinophils, on a cell for cell basis. None of the cells exhibited larvicidal activity in the absence of serum and serum alone had no effect. The reduction in larval infectivity caused by neutrophils in the presence of fresh normal mouse serum (NMS) was only marginally less than that obtained using immune mouse serum (IMS), suggesting that complement, which is activated by the larvae via the alternative pathway and mediates the adherence of both cell types, was able to promote the larvicidal effect of these cells in vitro. In contrast to neutrophils, eosinophils were considerably less effective in NMS than in IMS. Both NMS and IMS were ineffective if they had been heat-inactivated or incubated with methylamine at pH 8.0 to destroy complement activity. The immunoglobulin fraction of IMS was also ineffective in promoting neutrophil or eosinophil-mediated larval damage. These results indicate that in this in vitro system antibodies are incapable of directing the activity of either cell type in the absence of complement. A novel finding of this study was that neutrophils from uninfected mice were unable to reduce larval infectivity in the presence of fresh NMS or IMS. 'Altered' neutrophils possessing larvicidal activity appeared in the blood of mice within 4 days of infection with N. dubius.

Trypanosoma lewisi: the effect of thymectomy on the production of ablastin and the termination of the parasitaemia in infected rats.

This study investigated the effect of the depletion of T lymphocytes on the production of ablastin and the termination of the parasitaemias in Porton strain rats infected with Trypanosoma lewisi. Weanling rats were thymectomised or sham-thymectomised and then X-irradiated. After allowing 96 days for recovery from these procedures the rats were infected with T. lewisi. The rats were considered to be T lymphocyte depleted if they were unable to mount an antibody response to sheep red blood cells before the infection, a delayed type hypersensitivity response to trypanosomal antigen after they had cleared the infection, and if they succumbed to subsequent challenge with an intracellular bacterial parasite, Salmonella typhimurium C5. The T lymphocyte depleted rats were found to have no macroscopic thymus remnants visible at autopsy. The sham-thymectomised and unoperated control rats retained all the T lymphocyte functions tested for. There was no difference between the control and the T lymphocyte depleted rats in the ability to eliminate the parasite following infection with T. lewisi. Ablastin titres in the serum, measured by a sensitive in vitro assay, were found to be the same in each of the groups of rats tested, regardless of their T lymphocyte status. It is concluded that T lymphocytes are not essential for rats to eliminate a T. lewisi infection, nor for the production of ablastin.

Adherence of murine peripheral blood eosinophils and neutrophils to the different parasitic stages of Nematospiroides dubius.

Eosinophils and neutrophils, purified by density gradient centrifugation from the blood of infected mice resistant to reinfection, were tested for their ability to adhere to the different parasitic larval stages of the murine nematode parasite Nematospiroides dubius. Cells were tested for adherence to larvae which had been sensitised with immune mouse serum (IMS) or normal mouse serum (NMS) in the presence of CA2+ and Mg2+ ions. EDTA, or EGTA. Differences were observed in the degree of cell adherence to the different stages of the parasite. However, the adherence of the two cell types to any given stage of the parasite was similar. Adherence to the sheathed infective third-stage (L3) larvae, 96 h post-infective larvae and to adult worms depended to a large degree on conditions suitable for complement activation (viz. fresh serum and the presence of Ca2+ and Mg2+ ions). Complement was activated both via the alternative pathway by the parasite itself and via the classical pathway by parasite-bound antibodies. In these conditions, cell adherence probably occurred predominantly through the interaction of leucocyte third component of complement (C3) receptors with parasite-bound C3. In contrast, adherence of cells to exsheathed L3 and to the 48 h and 72 h post-infective larval stages appeared to involve antibody/Fc receptor as well as C3/C3 receptor interaction. The data indicate that N. dubius may undergo a series of antigenic changes during its life cycle and that antibodies capable of mediating granulocyte attachment are elicited predominantly against the early tissue developmental forms of the parasite.

The effect of splenectomy on the production of ablastin and the termination of the parasitaemia in rats infected with Trypanosoma lewisi.

This study investigated the effect of splenectomy on the production of ablastin and the elimination of Trypanosoma lewisi from the circulation of infected Porton rats. Rats were splenectomized or sham-splenectomized either before or at various stages during the infection and the course of the parasitaemia compared with untreated control animals. Ablastin titres in the serum were measured by a sensitive in vitro assay and were found to be the same in all the rats, regardless of the nature or timing of the operation. Splenectomized animals eliminated the parasite more slowly from the circulation than either the sham-splenectomized animals or unoperated controls. It is concluded that the presence of the spleen enables the rat to clear the parasite more rapidly from the circulation, but that the spleen is not essential for the production ablastin during the infection.

Haemagglutinin levels in haemolymph from the colonial ascidian Botrylloides leachii following injection with sheep or chicken erythrocytes.

Haemolymph was collected from colonies of Botrylloides leachii killed at various times after injection with sheep or chicken erythrocytes and the agglutination titre for sheep, chicken and guinea-pig erythrocytes was determined. Controls included haemolymph from groups of uninjected colonies and from others injected with balanced salt solution. Animals given a single injection of erythrocytes exhibited no change in titre until the second week when a marginal (2- to 4-fold) but statistically significant rise in titre was detected using each of the 3 types of indicator erythrocytes. A return to control values was observed by 6 weeks. The response to a second injection of erythrocytes, given 6 weeks after the first, appeared to be identical in magnitude and time of onset to the primary response, i.e. there was no indication of an anamnestic response. The increased haemagglutinating activity apparent 2 weeks after each injection was attributable to a rise in the HA-2 agglutinin specifically, since the activity of the HA-1 agglutinin (a calcium-dependent lectin which binds to guinea-pig erythrocytes only) remained unchanged. The results are considered in relation to self: non-self recognition in this species.

Properties of ablastin--a factor in the serum of rats infected with Trypanosoma lewisi which inhibits the parasites' division.

The present study investigated the nature of ablastin, a factor present in the serum of rats infected with Trypanosoma lewisi and which inhibits the parasites' division. Ablastin was unstable to dialysis at pH 1X8, was not adsorbed from serum by trypanosomes and could not be induced in vivo by means other than a natural infection. Attempts to purify ablastin from serum by conventional chromatographic techniques were unsuccessful. Removal of over 90% of the immunoglobulins from ablastinic serum did not reduce the ablastin titre. It is concluded that ablastin is unlikely to be an immunoglobulin as has been previously suggested.

Identification of the HA-2 agglutinin in the haemolymph of the ascidian Botrylloides leachii as the factor promoting adhesion of sheep erythrocytes to mouse macrophages.

The HA-2 agglutinin purified from B. leachii haemolymph is shown to mediate the binding of sheep erythrocytes to mouse macrophages. This agglutinin appears to be wholly responsible for the adhesive activity of haemolymph, since upon chromatography of the latter through Sephadex G-200, the adhesive activity for both sheep and guinea pig erythrocytes was confined to those fractions containing the HA-2 agglutinin. The HA-1 agglutinin recovered in earlier fractions was unable to promote the adhesion of guinea pig erythrocytes to macrophages. Adsorption experiments indicated that this was due to a lack of ligand sites on the macrophage surface. These data support earlier conclusions that the HA-1 and HA-2 agglutinins have distinct binding specificities.

An agglutinin in the haemolymph of an ascidian promoting adhesion of sheep erythrocytes to mouse macrophages.

Sheep erythrocytes sensitized with subagglutinating doses of haemolymph from Botrylloides leachii were found to bind strongly to mouse peritoneal macrophages in vitro. Erythrocytes sensitized with similar agglutinating doses of plant lectins or haemolymph from the fresh water crayfish Cherax destructor did not adhere. The adherence promoted by B. leachii haemolymph appears to be a heteroagglutination reaction mediated by a molecule which binds to lactose-like determinants on both erythrocytes and macrophages.

Identification of two carbohydrate-specific erythrocyte agglutinins in the haemolymph of the protochordate Botrylloides leachii.

The haemolymph of the colonial ascidian Botrylloides leachii contains two haemagglutinins which have been termed HA-1 and HA-2 respectively. The HA-1 agglutinin is specific for guinea-pig erythrocytes, requires Ca2+ ions for its activity and has an apparent mol. wt. of about 200,000. It can be adsorbed by guinea-pig but not human, sheep, mouse or pigeon erythrocytes. The agglutination of guinea-pig cells is reversibly inhibited by various mono- and oligosaccharides, lactose and D-galactose being among the most effective of those tested. The HA-2 agglutinin is smaller (mol.wt. approximately 63,000) and agglutinates a variety of erythrocyte types, including those from the human, guinea-pig, mouse, sheep, pigeon and chicken. The agglutination of all these cells is inhibited by lactose. Data from sugar-inhibition and adsorption experiments indicate that the HA-2 agglutinins are homogeneous with respect to binding site specificity. Divalent cations are not required for activity.

Alternative pathway activation of complement by a murine parasitic nematode (Nematospiroides dubius).

The cuticular surface of the infectious third-stage larvae of Nematospiroides dubius activates complement via the alternative pathway. Sensitisation of larvae with complement or with antibodies from the serum of immune mice (resistant to reinfection) promoted the adherence of mouse peritoneal exudate cells to the larval cuticle during incubation in vitro. The infectivity of larvae sensitized with antibody or complement was significantly reduced after incubation with cells from immune mice.