Hedvig Perlmann

Contactual lysis of antibody-coated chicken erythrocytes by purified lymphocytes

Abstract 51Cr-labeled chicken erythrocytes were lysed within 1–2 days when incubated with lymphocytes from human peripheral blood in the presence of heat-inactivated rabbit anti-chicken erythrocyte serum. Presence of nonlymphocytic leukocytes enhanced the initial rate of the reaction. However, final lysis produced by highly purified lymphocytes in the presence of antibody was usually more complete than that produced by polymorphonuclear leukocytes and monocytes. Lysis by purified lymphocytes was easily produced when the erythrocytes were pretreated with antiserum or when antiserum at dilutions up to 10−8 was incorporated in the incubation mixture. Pretreatment of the lymphocytes with antiserum was much less efficient. Lysis was not due to conventional activation of hemolytic complement. Induction of cytotoxicity seemed to require contact between lymphocytes and antibody, or antigen-antibody complexes on the erythrocytes. IgM-antibodies to Forssman antigen on the chicken erythrocyte surface were not active in this system. Although erythrophagocytosis by lymphocytic cells occurred, it was of minor importance for the cytotoxic reaction. The lymphocytes were not inactivated by contact with antibody-coated erythrocytes. No stable cytotoxic mediators, lytic for antibody-free erythrocytes, were released into the incubation medium from lymphocytes exposed to antibody-coated erythrocytes. The data provide strong evidence that the lytic steps of the reaction also required close contact between effector cells and target cells.

Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells.

During the past decade, experimental evidence has been brought forward in many laboratories that lymphocytes are capable of destroying appropriate target cells in vitro. These cytotoxic effects of lymphocytes have been assumed to reflect their effector role in tissue damaging immune responses such as occur in allograft rejection, tumor surveillance and certain autoimmune diseases (Perlmann & Holm 1969). Lymphocyte mediated cytotoxicity in vitro is complex and may involve a number of different pathways. The contention that a cytotoxic reaction is lymphocyte induced does not necessarily imply that the lymphocytes which start the reaction also are the cytotoxic effector cells, nor that the latter are lymphocytes at all. In addition, even in those instances in which there is evidence that both inducerand effector cells are lymphocytic, lymphocytes of different origin and function may participate in the cytotoxic reaction. Work with mice in alloimmune situations, involving the H2 system, has provided evidence that thymus derived lymphocytes (T-cells) are required for cytotoxicity (Blomgren et at. 1970, Cerottini et al. 1970a,b, Golstein et al. 1972a). In a few instances available data suggest that T-cells may be both inducerand effector cells (Golstein et al. I972a,b). Recent evidence also indicates that humoral antibodies may induce cytotoxicity of thymus-independent lymphoid cells (Harding et al. 1971, Van Boxel ct al.

IgE elevation and IgE anti-malarial antibodies in Plasmodium falciparum malaria; association of high IgE levels with cerebral malaria

In the course of studying immunoregulation in human Plasmodium falciparum malaria we have investigated IgE levels and IgE anti‐plasmodial antibodies in children and adults from areas of high malaria endemicity in both Africa and Asia. On average, 85% of all donors had significantly elevated levels of total IgE. A fraction of the IgE had anti‐plasmodial activity as revealed by ELISA with lysates of infected erythrocytes as antigen. Using synthetic peptides representing antigenic regions of two major plasmodial blood stage antigens, IgE antibody concentrations ranged from 5 to 15 ng/ml serum for each of the peptides. On average, the concentrations of the corresponding IgG antibodies were x500‐I000 higher. Immunoblotting of parasite lysates showed that most donors had IgE antibodies against one or several of a restricted number of plasmodial polypeptides, with antibodies against an antigen of mol. wt 45 kD already being present in all donors at an early age. Donors having IgE antibodies to particular antigens also frequently had corresponding IgG4 antibodies, reflecting underlying IL‐4‐dependent cellular mechanisms controlling formation of these isotypes. As infection with other parasites such as helminths is known lo induce IgE elevation, the results do not prove that plasmodial infections were the primary cause of IgE induction. However, the importance of plasmodial infection for IgE elevation was supported by the finding of significantly higher levels of IgE., but not of IgG, in children with cerebral malaria compared with patients with uncomplicated disease.

Dissection of the human antibody response to the malaria antigen Pf155/RESA into epitope specific components

The development of vaccines is presently receiving major attention in malaria research. As it is not possible to base malaria vaccines on the use of killed or attenuated organisms, the vaccines which are being developed are subunit vaccines in which the immunogens consist of defined parasite antigens or antigenic fragments. Since protective immunity to malaria involves both antibody-dependent and antibody-independent mechanisms, the immunogens in a subunit vaccine must have the capacity to induce relevant B- and T-cell responses in the majority of vaccinees. In turn, this requires good knowledge of these responses in humans who have acquired immunity through natural infection. In this paper we have summarized our recent work on the dissection into epitope-specific components of the human antibody response to the Plasmodium falciparum antigen Pf155/RESA, a recognized candidate for a vaccine against the asexual blood stages of this parasite. Epitope mapping of the antigen by means of short synthetic peptides led to the identification in several molecular regions of short amino acid sequences constituting linear and probably immunodominant B-cell epitopes. The antigenically most active region was located in the C-terminus of the molecule. This region, which consists of approximately 40 related, 4- or 8-amino acid long repeats, induced higher antibody concentrations in a larger number of malaria-immune donors than any of the other regions. A large fraction of these antibodies bound to short synthetic peptides representing the major repeat motifs of Pf155/RESA. Although these repeats are made up of closely related amino acid sequences, the antibody response to them was highly polyclonal, indicating the presence of several linear and probably also conformational epitopes which gave rise to a variety of cross-reacting as well as monospecific antibodies. Further analysis revealed that the levels of antibodies differing in specificity and/or avidity for different peptides varied independently of each other in individual donors. In an area (Liberia) where malaria transmission is holoendemic and perennial, these antibody profiles remained constant when individual donors were followed over several years. Since the C-terminal repeat region of Pf155/RESA is conserved in different P. falciparum strains, the results reflect differences in the genetic regulation of epitope-specific host responses rather than antigenic differences between infecting parasites. In donors living in an area with high but seasonal malaria transmission, antibody levels usually drop to lower levels when there is no transmission.(ABSTRACT TRUNCATED AT 400 WORDS)

IgE and tumor necrosis factor in malaria infection

IgE, the immunoglobulin instrumental in atopic diseases is also elevated in many infections. This paper reports on the occurrence and possible pathogenic role of IgE in human Plasmodium falciparum malaria, one of the most widely spread and severe infectious diseases world wide. Plasmodial infections induce IgE elevation in the blood of the majority of people living in malaria endemic areas and up to 5% of this IgE constitutes anti-malaria antibodies. Production of IgE is controlled by T cells and elevated IgE concentrations in the blood of malaria patients are the result of an increased ratio of T-helper 2 (Th2) over T-helper 1 (Th1) cells. The underlying Th1 to Th2 switch is controlled by a variety of environmental and genetic factors. The importance of the latter is demonstrated by the IgE levels occurring in monozygotic or dizygotic twins originating from malarious areas of Africa. While these levels were indistinguishable within monozygotic twin pairs, they were different within the dizygotic pairs. Comparison of the levels of total IgE or IgE anti-malaria antibodies in patients with uncomplicated malaria with those in patients with the severe form of the disease (cerebral malaria or severe malaria without cerebral involvement) indicate that these levels are significantly higher in the cases with severe disease. This is the reverse with IgG and suggests that IgE plays a role in malaria pathogenesis. An important pathogenic mediator causing malaria fever and tissue lesions is tumor necrosis factor (TNF), generally believed to be induced by toxins released from the parasite. However, sera from malaria patients can also cause TNF release from monocytes in a reaction dependent on the presence of IgE containing immune complexes or aggregates. This results in induction and cross-linking of Fcepsilon receptor II (CD23) and by binding to and activating these cells, IgE will contribute to a local over-production of TNF in capillaries and post-capillary venules where P. falciparum parasites or their products accumulate in the severe forms of this disease.

Cytotoxic Effects of Leukocytes Triggered by Complement Bound to Target Cells

Chromium-51-labeled chicken erythrocytes (E), treated with rabbit anti-Forssman antibody (A) and the first four (C1-4) or the first seven (C1-7) components of human complement (C), released isotope upon exposure to human leukocytes. Isotope release from EACJ-7 cells proceeded more rapidly and was more extensive than that from EACI-3 cells. Lysis of these cells was suppressed by pretreatment of leukocytes with antimycinA. Monocyte-enriched leukocyte preparations affected both types of target cell-complement intermediates, whereas purified lymphocytes lysed EACI-7 cells but not EACI-3 cells.

Elevated plasma levels of IgE in Plasmodium falciparum-primed individuals reflect an increased ratio of IL-4 to interferon-gamma (IFN-γ)-producing cells

People living in Plasmodium falciparum‐endemic areas frequently have elevated levels of total as well as P. falciparum‐specific serum IgE. This study aimed at investigating whether the elevated serum IgE levels reflect a shift in the balance between CD4+ T helper 1 (Th1) and T helper 2 (Th2) cells in individuals naturally exposed to the P. falciparum parasite. To investigate the role of Th1 and Th2 cells in the human P. falciparum system we used the ELISPOT assay to determine the ratio of IFN‐γ‐ and IL‐4‐producing cells after specific antigen or mitogen activation in vitro. The donors were individuals who had acquired immunity through natural exposure to the parasite. In response to the specific malaria antigens, very few IL‐4‐producing cells were seen. However, in the response of individual donors to the polyclonal T cell activator, leucoagglutinin (La), the anti‐malarial IgE levels in plasma were correlated with an increased ratio of IL‐4/IFN‐γ producing cells. Thus, donors with ratios of IL‐4/IFN‐γ > 1 exhibited mean plasma anti‐malarial IgE levels significantly greater than those with ratios < 1. In individuals not living in P. falciparum‐endemic areas the ratio of IL‐4/IFN‐γ was always < 1. Taken together, our data suggest a shift in the balance between Th1 and Th2 cells in naturally P. falciparum‐primed individuals, associated with elevated anti‐P. falciparum plasma IgE levels. The role and biological significance of IgE (Th2‐type immune response) for protection against P. falciparum and/or pathogenesis of malaria require further study.

Allelic polymorphisms in the repeat and promoter regions of the interleukin-4 gene and malaria severity in Ghanaian children

Immunoglobulin E has been associated with severe malaria suggesting a regulatory role for interleukin (IL)‐4 and/or IgE in the pathogenesis of severe malaria. We have investigated possible associations between  polymorphisms  in  the  IL‐4  repeat  region  (intron  3)  and  promoter  regions  (IL‐4 +33CT  and − 590CT) in Ghanaian children with severe malaria. There was a significantly higher frequency of IL‐4 intron‐3 B1B1 genotype in the cerebral malaria group [P < 0·0001, odds ratio (OR) = 8·7]. The genotype and allele frequencies of the IL‐4 –590 and +33 polymorphisms did not differ between the four study groups. Carriers of IL‐4 +33T/−590T with cerebral malaria had elevated total IgE compared to non‐carriers (P = 0·03). Our data suggest that IL‐4 and/or IgE play a regulatory role in the pathogenesis of severe or complicated malaria.

Cytotoxic Action of Stimulated Lymphocytes on Allogenic and Autologous Erythrocytes

Fowl erythrocytes are lysed when exposed to an excess of fowl blood lymphocytes in the presence of phytohemagglutinin. No significant cell damage is seen in the absence of phytohemagglutinin, or when the lymphocytes are replaced by malignant lymphoid cells, thymus cells, or nonlymphoid cells. The lymphocytes remain viable during the reaction. Differences in histocompatibility between lymphocytes and erythrocytes are not required. Autologous lymphocytes are cytotoxic to the same extent as allogenic lymphocytes over a wide range of experimental conditions.

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

Abstract We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.