Activity of monocytes of subjects sensitized to Plasmodium falciparum: adherence and phagocytosis of merozoites in vitro. Role of cytophylic antibodies

Peripheral blood monocytes from subjects immune to malaria are able to ingest more efficiently P. falciparum merozoites under in vitro conditions than those from non-immune subjects. Similar results were recorded when using monocytes from healthy controls preincubated with sera from immune individuals or IgG prepared from these sera. The phagocytic process does not seem to be strain specific. In the conditions of our assay, no phagocytosis of parasitized erythrocytes was observed. The main target of the IgG armed monocyte is the merozoite.     

Plasmodium falciparum: surface modifications of infected erythrocytes from clinical isolates

 Infections of human erythrocytes with the mature asexual blood stages of Plasmodium falciparum result in antigenic changes in the host cell membrane that, by virtue of their position, length of exposure, and close association with functional changes critical to pathogenesis, are a potential important target for host effector mechanisms. These parasite-induced antigens expressed on the surface of infected erythrocytes have been shown to exhibit considerable polymorphism. An antibody-mediated agglutination assay using malaria serum samples from different regions of Venezuela has been developed to examine the extent of antigenic diversity of infected red blood cells (IRBC) taken from subjects with naturally acquired P. falciparum infections. An important humoral immune recognition of surface molecules from red blood cells infected with a wide variety of clinical isolates of P. falciparum was observed even when sera from individuals experiencing a single episode of malaria were used. A process of in vivo antigenic variation of surface molecules is postulated, since agglutination of IRBC was observed with acute heterologous but not autologous sera. When sera obtained from Amerindians inhabiting the Venezuelan Amazon were assayed, a strong immune response to different parasite isolates, including those of another geographic region, was observed, suggesting the recognition of highly conserved immunogenic parasitic epitopes in people exposed to multiple malaria infections. Received: 20 June 1995 / Accepted: 3 November 1995     

Independence of complement on in vitro immune phagocytosis of plasmodium falciparum parasitised erythrocytes by human monocytes and polymorphonuclear leukocytes

Monocytes and polymorphonuclear leukocytes from normal blood donors phagocytosed preferentially Plasmodium falciparum-infected red blood cells (IRBC) in presence of sera from individuals living in areas endemic for malaria. Total complement or factor B heat inactivation of immune or normal serum does not alter opsonic activity directed against IRBC.     

Enhanced Plasmodium falciparum merozoite phagocytosis by monocytes from immune individuals.

Phagocytosis of merozoites and schizont-infected erythrocytes prepared from continuous cultures by peripheral blood monocytes from patients with falciparum malaria was investigated with an in vitro assay. Monocytes do ingest merozoites of Plasmodium falciparum, but rarely phagocytose parasitized or nonparasitized erythrocytes in the absence of immune serum. The monocytes from hyperimmune subjects were significantly more efficient in the ingestion of merozoites than were those obtained from sensitized or noninfected subjects. These data indicate, first, that the merozoite rather than the parasitized erythrocyte is the specific target for blood phagocytic cells in human falciparum malaria and, second, that the phagocytosis of merozoites by peripheral blood monocytes increases depending on the level of specific immunity.     

Babesia bovis: In vitro phagocytosis promoted by immune serum and by antibodies produced against protective antigens

The in vitro phagocytosis of bothBabesia bovis-infected red cells and of parasites exposed by lysis of infected red blood cells is demonstrated in a phagocytic mouse model. Twenty-fourB. bovis immune sera were tested alone or as a pool as were antibodies (DS antibodies) raised against aB. bovis protective fraction, prepared by dextran sulfate precipitation. All the immune sera failed to promote significant levels of phagocytosis, whereas the other antibodies (DS antibodies) consistently induced phagocytosis of infected cells in all the experiments carried out. This study shows that antibody specificity is critical to the opsonization of infected red cells and parasites during in vitro phagocytosis and suggests that phagocytosis is one of the mechanisms in the in vivo immune response againstBabesia species.     

Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes

A method is described for the visualization of red blood cells infected with Plasmodium falciparum ingested by monocytes or polymorphonuclear leukocytes (PMN) after in vitro incubation. Smears were stained with peroxidase followed by 4,6-diamino-2-phenylindole (DAPI) staining specific for DNA. Monocytes or PMN were identified under normal illumination by the peroxidase stain and the nuclei of these cells as well as the parasites were identified by means of the DAPI stain with ultraviolet light. Using this method we found that monocytes and PMN from normal blood donors preferentially phagocytose plasmodium falciparum infected red blood cells in the presence of sera from subjects living in areas endemic for malaria.     

Differences in human antibody reactivity to Plasmodium falciparum variant surface antigens are dependent on age and malaria transmission intensity in northeastern Tanzania

Plasmodium falciparum variant surface antigens (VSA) are involved in the pathogenesis of malaria. Immunoglobulin G (IgG) with specificity for VSA (anti-VSA IgG) is therefore considered important for acquired immunity. To better understand the nature and dynamics of variant-specific IgG responses at population level, we conducted an immunoepidemiological study in nearby communities in northeastern Tanzania, situated at different altitudes and therefore exposed to different levels of P. falciparum transmission intensity. Samples of plasma and infected red blood cells (IRBC) were collected from 759 individuals aged 0 to 19 years. Plasma levels of IgG with specificity for VSA expressed by a panel of different parasite isolates were measured by flow cytometry, while the ability of plasma to inhibit IRBC adhesion to CD36 was examined in cellular assays. The level and repertoire of the heterologous anti-VSA IgG response developed dramatically in individuals at 1 to 2 years of age in the high-transmission area, reaching a maximum level at around 10 years of age; only a modest further increase was observed among older children and adults. In contrast, at lower levels of malaria transmission, anti-VSA IgG levels were lower and the repertoire was more narrow, and similar age- and transmission-dependent differences were observed with regard to the ability of the plasma samples to inhibit adhesion of IRBC to CD36. These differences indicate a strong and dynamic relationship between malaria exposure and functional characteristics of the variant-specific antibody response, which is likely to be important for protection against malaria.     

Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: functional role of IgG subclasses.

A phagocytic assay performed with human peripheral mononuclear cells and malaria-infected erythrocytes enabled the study of opsonizing antibodies in human serum from donors presenting different levels of protective immunity. Opsonizing activity was found in sera from individuals who could be considered immune, i.e. asymptomatic parasite carriers and subjects residing in endemic regions who presented neither symptoms nor parasites. This contrasted with subjects showing an absence of symptoms or who had experienced only a single malarial infection. All sera contained high levels of antimalarial antibodies, as shown by immunofluorescence assay (IFA). IgG of different subclasses were immunopurified from these sera. All subclasses were shown to bind to the surface of infected erythrocytes by FACS analysis, but only IgG1 and IgG3 were able to mediate opsonization. IgG2 and IgG4 did not opsonize and inhibited the opsonizing activity of IgG1 and IgG3 in competition experiments. These results suggest the existence of a correlation between immune protection, the ability of serum to mediate opsonization of infected erythrocytes and the predominance, in this serum, of IgG1 and IgG3 over IgG2 and IgG4 directed against the surface of the infected erythrocytes.     

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.     

Opsonization as an effector mechanism in human protection against asexual blood stages of Plasmodium falciparum: Functional role of IgG subclasses

A phagocytic assay performed with human peripheral mononuclear cells and malaria-infected erythrocytes enabled the study of opsonizing antibodies in human serum from donors presenting different levels of protective immunity. Opsonizing activity was found in sera from individuals who could be considered immune, i.e. asymptomatic parasite carriers and subjects residing in endemic regions who presented neither symptoms nor parasites. This contrasted with subjects showing an absence of symptoms or who had experienced only a single malarial infection. All sera contained high levels of antimalarial antibodies, as shown by immunofluorescence assay (IFA). IgG of different subclasses were immunopurified from these sera. All subclasses were shown to bind to the surface of infected erythrocytes by FACS analysis, but only IgG1 and IgG3 were able to mediate opsonization. IgG2 and IgG4 did not opsonize and inhibited the opsonizing activity of IgG1 and IgG3 in competition experiments. These results suggest the existence of a correlation between immune protection, the ability of serum to mediate opsonization of infected erythrocytes and the predominance, in this serum, of IgG1 and IgG3 over IgG2 and IgG4 directed against the surface of the infected erythrocytes.     

Immune elimination of aging platelets by autologous monocytes: role of membrane-specific autoantibody

Membrane-bound IgG was found only on old populations of platelets from normal individuals. This IgG could be dissociated from senescent cells by repeatedly heating the cells. Heat-eluted IgG (He-IgG) prepared from senescent red blood cells was capable of binding to either heat-treated old platelets or Vibrio cholerae neuraminidase (VCN)-treated young platelets, suggesting expression of a common age-dependent antigen on the senescent red blood cells and old platelets. We analyzed the role of membrane-bound IgG in the immune elimination of aging platelets by direct phagocytosis of different platelet subpopulations by autologous monocytes in vitro. While removal of He-IgG from old platelets inhibited their phagocytosis, preincubation of either heat-treated old or VCN-treated young platelets promoted phagocytosis of these cells by autologous monocytes. The phagocytosis of senescent cells required intact IgG on these cells. Either removal of Fc fragments from He-IgG or treatment of autologous monocytes with Fc fragments prior to the phagocytosis assay resulted in a marked reduction of phagocytosis (greater than 75%). We conclude that Fc receptors on the monocytes and the presence of membrane-specific IgG are crucial elements for immune elimination of senescent platelets.     

Fcgamma receptor-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes in vitro

Although convincing evidence exists for the role of immunoglobulin G (IgG) antibodies in immunity to malaria, antibody titres do not usually predict protection. In this study we have assessed the interaction between Plasmodium falciparum-infected erythrocytes (PE), opsonized with immune serum containing different amounts of IgG antibody isotypes, with either THP-1 cells, ex-vivo human monocytes or IIAI.6 transfectant cells expressing Fc(gamma)RIIa-Arg/Arg131 or -His/His131 allotypes. Our results show that PMA-treated THP-1 cells were capable of phagocytosing serum-opsonized PE by Fc(gamma)RI (CD64) and Fc(gamma)RIIa (CD32), acting synergistically. The known Fc(gamma)RIIa polymorphism motivated us to examine its influence on IgG isotype-mediated phagocytosis of opsonized PE with human monocytes and the IIAI.6 transfectant cells expressing either allelic forms. Regardless of the cell type, PE phagocytosis with Fc(gamma)RIIa-His/His131 was highest following opsonization with a predominantly IgG3-containing immune serum pool. In contrast, PE phagocytosis with Fc(gamma)RIIa-Arg/Arg131 tended to be higher with an IgG1-containing pool. These results suggest a genetically determined influence of effector cell phenotype on IgG antibody-pathogen interaction in P. falciparum malaria.     

Phagocytosis does not play a major role in naturally acquired transmission-blocking immunity to Plasmodium falciparum malaria

Phagocytosis of Plasmodium falciparum sexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26 degrees C, compared with 37 degrees C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.     

Relationship of binding of immunoglobulin G to Plasmodium falciparum-infected erythrocytes with parasite endemicity and antibody responses to conserved antigen in immune individuals

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.     

Immunology of streptokinase in human subjects.

Antigens of Plasmodium falciparum, in supernatants of in vitro cultures of the parasite were affinity purified on columns prepared with the IgG fraction of the serum of an immune individual. The purified antigens induced proliferation of lymphocytes from persons who had recently had malaria. The responses were strongest with lymphocytes from individuals infected with falciparum and ovale malaria; vivax malaria infections induced a lower level of response and lymphocytes of unsensitized individuals were little affected. Lymphocytes from unsensitized individuals did not respond to the affinity purified antigen preparations from malaria patients' sera indicating that significant amounts of non-specific mitogens were not present.     

Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys.

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 inhibit opsonization of infected erythrocytes by hyperimmune Saimiri sera, indicating that they contain target epitopes involved in the phagocytosis of infected erythrocytes. We have investigated in this study the immune response of Saimiri monkeys with previous experience of malaria infections (preimmune monkeys) after injection of these recombinant antigens, administered alone or simultaneously. The humoral response to the recombinant antigens was monitored by radioimmunoassay, and the response to P. falciparum blood stages was assayed by immunofluorescence. The relative proportion of protective versus nonprotective immunoglobulin subtypes was investigated by using 3A2/G6 and 3E4/H8 monoclonal antibodies, and the capacity of the antisera to promote in vitro phagocytosis of infected erythrocytes was evaluated. The antigens evoked in most cases a secondary-type antibody response, resulting in important increases in antigen-specific antibody titers and concomitantly in anti-P. falciparum titers. The ratio of 3A2/G6 to 3E4/H8 immunoglobulin subtypes varied with the immunogen used. Opsonizing antibodies were boosted in several animals, the most promising combination being the mixture of PfEB200 and R23 that induced long-lasting production in five of five animals. The detectable opsonizing activity appearing after immunization of the animals was antigen specific, as it was lost after adsorption of the recombinant antigens. The challenge of the animals with blood stage parasites confirmed previous findings showing a correlation between the presence of detectable opsonizing antibodies in serum and protection.     

Plasmodium falciparum: inhibition/reversal of cytoadherence of Thai isolates to melanoma cells by local immune sera.

The effect of sera on the cytoadherence in vitro of Plasmodium falciparum-infected erythrocytes to melanoma cells was examined. Sera from 19 healthy individuals living in endemic malarious areas in Thailand and 24 patients with P. falciparum malaria were tested against four local P. falciparum isolates. Out of 57 sera examined, 12 (21%) showed significant inhibition (greater than 50%) of cytoadherence for at least one isolate. Anti-malarial IgG antibody titres were determined for all 57 sera and although 11 of the 12 inhibitory sera had relatively high titres, 36 out of 47 sera with similarly high titres showed no significant inhibitory activity. Convalescent sera were no more effective than corresponding acute sera in inhibiting the cytoadherence of erythrocytes infected with any of the four heterologous isolates examined. Sera which significantly inhibited cytoadherence were also capable of reversing cytoadherence, and pooled plasma, from healthy individuals living in malarious areas, was effective in significantly reversing the in vitro cytoadherence of all the five parasite isolates examined. The results confirm the antibody mediated strain-specific nature of the inhibition of cytoadherence and stress the difficulty in selecting immune sera potentially useful for the immunotherapy of cerebral malaria patients in Thailand.     

Examination of Peripheral Blood Mononuclear Cells and Sera from Thai Adults Naturally Infected with Malaria in Assays of Blastogenic Responsiveness to Mitogenic Lectins and Allogeneic Cell Surface Antigens

We have previously observed that Thai adults who are infected with malaria have a loss of peripheral blood T cells, and that patient sera contain lymphocytotoxic antibodies. In the present study, we examined peripheral blood mononuclear cells from Thai adults naturally infected with Plasmodium falciparum and Plasmodium vivax for the capacity to undergo blastogenesis in response to phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cell surface antigens in a one-way mixed leukocyte reaction. In addition, sera from actively infected patients were examined with regard to suppressive capabilities toward normal lymphocyte blastogenesis by using the same assays. We found that patient mononuclear cells exhibited normal reactivity to phytohemagglutinin, concanavalin A, and pokeweed mitogen when compared with controls. However, peripheral blood mononuclear cells from patients had a decreased stimulatory capacity in the allogeneic mixed leukocyte reaction, and P. vivax, but not P. falciparum, lymphocytes exhibited decreased responsiveness in the mixed leukocyte reaction. Furthermore, sera from patients with active malaria induced decreased responsiveness by normal mononuclear cells to phytohemagglutinin and concanavalin A, but not pokeweed mitogen; pooled P. falciparum sera caused decreased responsiveness to allogeneic cell surface antigens in the mixed leukocyte reaction. These studies indicate that despite the lost of circulating T cells during the course of infection with malaria, blastogenic responsiveness remains intact, and that sera from patients with malaria are capable of exerting negative immunoregulatory effects.     

Comparative phagocytosis of cadmium microcrystals by peripheral blood glass-adherent cells of newborn and healthy adults

Microcrystals of cadmium, saturated with human serum albumin, were employed as a microassay for phagocytosis by glass-adherent blood cells. Using this assay, it was found that leukocytes of newborns possess significantly higher phagocytic activity and that a higher proportion of active phagocytic cells is present in newborns than in adults. In contrast, adult sera contain higher level of opsonins for cadmium microcrystals than cord sera. Heat inactivation and zymosan absorption of both adult and cord sera decreased significantly the opsonic power of sera, thus suggesting the participation of complement in opsonization of these particles.     

Anti-lymphocytotoxic antibodies in sera of Thai adults infected with Plasmodium falciparum or Plasmodium vivax.

Because of the potential for the elimination of lymphocytes through anti-lymphocytotoxic antibodies we examined individual sera of patients infected with falciparum or vivax malaria for the presence of antibodies against normal peripheral blood mononuclear cells. In assays done at 15 degrees C, 95% of the P. falciparum patients and 98% of the P. vivax patients showed evidence for antibody activity. Activity at 37 degrees C was significantly less than that at 15 degrees C. These studies suggest that infection with malaria induces anti-lymphocytotoxic antibodies which are predominantly cold-reactive. It is possible that this phenomenon plays a role in modulating the immune response of patients toward malaria.