IgE-dependent killing of Schistosoma mansoni schistosomula by human platelets: modulation by T cell products

The in vitro stimulation of T lymphocytes is known to induce the release of factors that possess distinct biological activities. In the present report, we describe the presence, in supernatants of Schistosoma mansoni antigen stimulated T cells from S. mansoni infected patients, of a factor able to inhibit the IgE-dependent platelet cytotoxicity of the same individuals toward the young larvae of S. mansoni.     

Increased IgE-dependent cytotoxicity by blood mononuclear cells of allergic patients.

Peripheral blood mononuclear cells from 14 healthy donors and 22 allergic patients were incubated with 51Cr-labelled chicken erythrocytes coated with an IgE myeloma protein or rabbit IgG antibodies. Mononuclear cells from patients with severe atopic disorders released a significantly greater percentage of 51Cr (P less than 0.001) from IgE-coated target cells than mononuclear cells from healthy controls, patients with mild atopic disease, or patients with severe atopic disease taking oral prednisone. Specific 51Cr-release from IgE-coated target cells was directly correlated to the percentage of monocytes (latex-ingesting cells) with Fc receptors for IgE (r = 0.87, P less than 0.01) as detected by a rosette assay employing ox erythrocytes coated with IgE. Mononuclear cells from patients and normals released similar amounts of 51Cr from IgG-sensitized target cells. Depletion of monocytes from mononuclear cell preparations from two severe atopic patients decreased 51Cr-release from IgE-coated target cells to levels seen in healthy donors or patients with mild allergic disease. These results demonstrate that mononuclear cells from severely allergic patients have a significantly increased cytotoxicity toward IgE-coated targets coated target cells and that this cytotoxicity correlates highly with the percentage of monocytes with Fc receptors for IgE in these mononuclear preparations.     

Inhibition of human eosinophil chemotaxis and of the IgE-dependent stimulation of human blood platelets by cetirizine

Cetirizine is a new anti-allergic compound with a potent, long-acting, and specific antihistaminic property. Strongly active in the therapy of urticaria and seasonal or perennial rhinitis, it has been shown to inhibit the in vivo eosinophil attraction at skin sites challenged with allergen in atopic patients. In the present work, we confirmed that, at a therapeutical concentration, this molecule had a potent inhibitory action in vitro on eosinophil chemotaxis induced either by N-formyl-Met-Leu-Phe or platelet-activating factor and also on the IgE-dependent stimulation of platelets. These observations appear in favour of a possible role for cetirizine in the modulation of inflammatory cell interactions in allergic processes.     

IgE-dependent cellular adhesion and cytotoxicity to Litomosoides carinii microfilariae--nature of effector cells.

Albino rats immunized with sonicated microfilarial antigen incorporated in Freund's complete adjuvant, produce antibodies that promote cell-mediated adhesion and killing of Litomosoides carinii microfilariae in vitro. Using highly purified cell populations, it has been shown that macrophages and neutrophils are most active in this phenomenon. Eosinophils, while adhering readily to parasites in the presence of the antibody, did not affect the viability of the parasites when observed after 24 hr incubation.     

IgE-dependent adherence and cytotoxicity of rat spleen and peritoneal cells to Litomosoides carinii microfilariae.

Serum taken after the termination of microfilaraemia from rats infected with the filarial parasite Litomosoides carinii brought about adherence and cytotoxicity of normal rat spleen and peritoneal cells to microfilariae. The activity could be absorbed to, and eluted from, anti-rat IgE, but not anti-rat IgG, immunosorbent columns. Immune serum heated to 56 degrees C for 3 hr did not cause cellular adherence or cytotoxicity; the addition of fresh normal rat serum failed to restore activity. Fresh rat serum did, however, restore activity to immune serum which was inactive after being heated to 56 degrees C for 30 min. EDTA, EGTA and diethylcarbamazine inhibited adherence. It is concluded that IgE antibodies are responsible for cellular adherence and cytotoxicity and that complement may play a part, as yet undefined, in these reactions.     

Quantitation of eosinophil Major Basic Protein cytotoxicity to rodent respiratory epithelium

Eosinophil Major Basic Protein (MBP) may be a potent effector in damaging airway epithelium and inducing acute (2–3 h) hyperresponsiveness to agonists in primates. Accordingly, interactions between human eosinophil MBP and guinea-pig airway epithelium were quantitated biochemically. MBP was extracted from human eosinophils and purified by size-exclusion HPLC. This resulted in a single protein band on electrophoresis, which cross-reacted with antisera raised to peptides derived from the predicted sequence of human MBP. This human MBP caused modest, but statistically significant, damage to respiratory epithelium (16.4% increase in efflux of⁵¹Cr from guinea-pig tracheal rings) after 3 h of incubation with 10^–4 M concentration, but not with lower concentrations. These data demonstrates that MBP cytotoxicity to intact epithelium can be rapidly measuredin vitro, and suggests that rodent airway epithelium may be relatively resistant to the cytotoxic effects of MBP.     

Enhancement of spontaneous killer cytotoxicity by soluble factor

Two-stage stimulation of SK activity of normal human peripheral blood lymphocytes by soluble factors was demonstrated. The first stage was initiated by factors present in supernatant derived from normal B-LCL cultures. Only cell lines which could induce SK activity in culture in an MLR-type reaction had the capacity to produce the active factor. Supernatant factor required adherent cells to cause SK augmentation. The interaction of adherent cells plus supernatant factor resulted in the production of a second soluble factor which stimulated an increase in SK activity in responding lymphocyte populations. This second stage involved a different soluble factor which acted directly on the non-SK, Fc-negative lymphocyte population, and within 3 hr. Data obtained using antisera to interferon (IF) indicated that IF is a component of the second soluble factor, and not of the supernatant factor derived from the B-LCL.     

Identification and measurement of rat eosinophil phospholipase D. Its activity on schistosomula phospholipids

A sensitive assay, using [¹⁴C]lecithin as a substrate, has been developed for the measurement of phospholipase activity in rat peritoneal polymorphonuclear leukocytes. Cell extracts were found to contain a phospholipase D activity and indirect evidence suggested that eosinophils are responsible for the cleavage of lecithin. Intact peritoneal cells were also able to hydrolyze exogenous [¹⁴C]lecithin in vitro. When [³H]choline-labeled schistosomula were used as targets in antibody-dependent cytotoxicity experiments, the radioactivity of lecithin decreased more rapidly in a complete cytotoxicity system than in controls, suggesting that hydrolysis of schitosomula phosholipids occured during the killing process.     

Impairment of protective immunity to blood-stage malaria by concurrent nematode infection

Helminthiases, which are highly prevalent in areas where malaria is endemic, have been shown to modulate or suppress the immune response to unrelated antigens or pathogens. In this study, we established a murine model of coinfection with a gastrointestinal nematode parasite, Heligmosomoides polygyrus, and the blood-stage malaria parasite Plasmodium chabaudi AS in order to investigate the modulation of antimalarial immunity by concurrent nematode infection. Chronic infection with the nematode for 2, 3, or 5 weeks before P. chabaudi AS infection severely impaired the ability of C57BL/6 mice to control malaria, as demonstrated by severe mortality and significantly increased malaria peak parasitemia levels. Coinfected mice produced significantly lower levels of gamma interferon (IFN-gamma) during P. chabaudi AS infection than mice infected with malaria alone. Concurrent nematode infection also suppressed production of type 1-associated, malaria-specific immunoglobulin G2a. Mice either infected with the nematode alone or coinfected with the nematode and malaria had high transforming growth factor beta1 (TGF-beta1) levels, and concurrent nematode and malaria infections resulted in high levels of interleukin-10 in vivo. Splenic CD11c(+) dendritic cells (DC) from mice infected with malaria alone and coinfected mice showed similarly increased expression of CD40, CD80, and CD86, but DC from coinfected mice were unable to induce CD4(+) T-cell proliferation and optimal IFN-gamma production in response to the malaria antigen in vitro. Importantly, treatment of nematode-infected mice with an anthelmintic drug prior to malaria infection fully restored protective antimalarial immunity and reduced TGF-beta1 levels. These results demonstrate that concurrent nematode infection strongly modulates multiple aspects of immunity to blood-stage malaria and consequently impairs the development of protective antimalarial immunity.     

Enhancement of eosinophil leukocyte chemotaxis by a factor in normal human serum (SEF).

An activity in normal human serum is described which enhances the migration of eosinophils towards neutrophil-derived eosinophil chemotactic factor (ECF). The serum-enhancing factor (SEF), in contrast to the inhibitors in serum, is expressed primarily during weak chemotaxis, acts in a cell-directed fashion, increases the chemokinesis of eosinophils and, in the presence of ECF, enhances chemotaxis in a synergistic fashion. SEF is found in sera of several mammalian species, affects human and guinea pig eosinophils, and it is heat-labile (56 degrees C). On Sephadex column chromatography, SEF has a molecular weight of approximately 800,000 daltons. In some sera, a second activity with a molecular weight of approximately 200,000 daltons is eluted. SEF does not enhance eosinophil migration once the cells have been deactivated in vitro by their chemotactic factor. Because of its wide distribution, SEF may play an important modulating role both in in vitro assay systems and in vivo.     

IL-3 is required for increases in blood basophils in nematode infection in mice and can enhance IgE-dependent IL-4 production by basophils in vitro

Basophils represent potential effector and immunoregulatory cells, as well as a potential source of IL-4, during the immune response elicited by infection with the nematode Nippostrongylus brasiliensis (N.b.), and in other settings. However, the factors which regulate the numbers of blood basophils in mice, or the ability of these cells to produce IL-4, are not fully understood. We found that infection of mice with the nematodes N.b. or Strongyloides venezuelensis (S.v.) induced substantial increases in the numbers of blood basophils (to as high as 18% of circulating blood leukocytes). Experiments in IL-3-/- vs IL-3+/+ mice, and in IL-3-treated IL-3-/- mice, showed that essentially all of the increases in blood or bone marrow basophils during N.b. or S.v. infection were IL-3 dependent. Many of the blood, bone marrow or liver-derived basophils from IL-3-/- or IL-3+/+ mice expressed intracellular IL-4 upon stimulation with anti-IgE in vitro. However, after incubation of the cells with exogenous IgE in vitro, blood- or liver-derived basophils from IL-3+/+ mice exhibited higher levels of intracellular IL-4 after stimulation with anti-IgE than did basophils derived from IL-3-/- mice. Thus, IL-3 is a major regulator of the marked increases in blood basophil levels observed during infection of mice with N.b. or S.v. and also can enhance levels of intracellular IL-4 upon activation of basophils with anti-IgE in vitro.     

Reduced protective efficacy of a blood-stage malaria vaccine by concurrent nematode infection

Helminth infections, which are prevalent in areas where malaria is endemic, have been shown to modulate immune responses to unrelated pathogens and have been implicated in poor efficacy of malaria vaccines in humans. We established a murine coinfection model involving blood-stage Plasmodium chabaudi AS malaria and a gastrointestinal nematode, Heligmosomoides polygyrus, to investigate the impact of nematode infection on the protective efficacy of a malaria vaccine. C57BL/6 mice immunized with crude blood-stage P. chabaudi AS antigen in TiterMax adjuvant developed strong protection against malaria challenge. The same immunization protocol failed to induce strong protection in H. polygyrus-infected mice. Immunized nematode-infected mice produced significantly lower levels of malaria-specific antibody than nematode-free mice produced. In response to nematode and malarial antigens, spleen cells from immunized nematode-infected mice produced significantly lower levels of gamma interferon but more interleukin-4 (IL-4), IL-13, and IL-10 in vitro than spleen cells from immunized nematode-free mice produced. Furthermore, H. polygyrus infection also induced a strong transforming growth factor beta1 response in vivo and in vitro. Deworming treatment of H. polygyrus-infected mice before antimalarial immunization, but not deworming treatment after antimalarial immunization, restored the protective immunity to malaria challenge. These results demonstrate that concurrent nematode infection strongly modulates immune responses induced by an experimental malaria vaccine and consequently suppresses the protective efficacy of the vaccine against malaria challenge.     

Induction of delayed hypersensitivity by dinitrophenylated lymphocytes

Erythrocytes, serum proteins and both living and killed lymphocytes from the peripheral blood of guinea-pigs were dinitrophenylated and then injected intraperitoneally into either the donor or other guinea-pigs. Animals sensitized by intradermal DNCB and unsensitized normal guinea-pigs served as positive and negative controls respectively. Eleven to 14 days after injection, the animals were tested with topically applied DNCB and the intensity of the reactions assessed by the local exudation of bovine serum albumin labelled with radio-iodine ([¹³¹I]BSA.)

Sensitization was achieved with dinitrophenylated living autologous lymphocytes, to a lesser degree with homologous lymphocytes, but not with erythrocytes, serum proteins or killed lymphocytes.

     

The response of histamine degrading enzymes to nematode infection

The response of intestinal mucosal enzymes which metabolize histamine i.e. diamine oxidase (DAO), histamine N-methyltransferase (HMT), and monoamine oxidase (MAO), to infection withNippostrongylus brasiliensis has been examined in mice and compared to the changes evoked byin vivo administration of compound 48/80. Infection with the parasite resulted in a significant decrease in the concentration of both amine oxidases, followed by recovery of MAO and an overshoot in DAO activity. HMT activity was enhanced at the beginning of infection, then decreased markedly by days 11 to 15, and sharply increased thereafter. Histamine levels were on average only 20% higher than the basal levels over the entire period studied, except on day 4 when they were slightly reduced. Histamine is alleged to be a potential inducing factor for degrading enzymes. Consistently, the histamine releaser 48/80 significantly elevated intestinal mucosal DAO and in some of the mice also increased HMT activity.Supported by Wpr 5 and CPBP 06.03.1.2.     

Enhancement of antibody-dependent cell-mediated cytotoxicity of herpesvirus-infected cells by complement.

An investigation was made of the effects of complement on the levels of antibody-dependence cytotoxicity (ADCC) mediated by bovine leukocytes against herpesvirus-infected target cells. Neutrophil-mediated ADCC was considerably enhanced upon the addition of low levels of complement that alone failed to induce lysis of antibody-sensitized target cells. This enhancement was most apparent under suboptimum conditions such as at low effector-to-target cell ratios, low levels of sensitizing antiserum, and short-duration assays. Furthermore, cells and classes of immunoglobulin unable to induce ADCC could do so in the presence of complement. The action of complement is considered in terms of a more tenacious bond formed between effector and target cells. The implications of the results are discussed in terms of the part that complement might play in enhancing antiviral recovery processes.     

Interleukin-12 promotes a chronic intestinal nematode infection

Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively. The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role. Interleukin (IL)-12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)-γ from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system. IL-12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania. It has also been proposed as an anti-tumor agent and for use in the treatment of HIV. Conversely, IL-12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity. Our studies demonstrate that by driving a strong Th1 response, IL-12 promotes chronic T. muris infection when given to normally resistant BALB/K mice. Parasite-specific IgG2a, a Th1 parameter of infection, was greatly up-regulated, whereas some Th2 parameters of infection were down-regulated. IL-12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response. The actions of IL-12 in promoting a chronic infection were IFN-γ dependent as an anti-IFN-γ mAb abrogated the effects of IL-12.     

Enhancement of human monocyte cytotoxicity by both interferon-gamma and -beta and comparison to other stimuli

Recombinant interferon preparations caused a dose-dependent increase of human monocyte cytotoxicity to the K562 and Daudi cell lines. Both rIFN-gamma and rIFN-beta enhanced this function to a similar extent, while rIFN-alpha c had less effect when compared on the basis of their anti-viral effects. Endotoxin and concanavalin A increased basal monocyte cytotoxicity while phagocytosis of latex particles had no effect. The increased monocyte cytotoxic effect of rIFN-beta was completely abrogated by monoclonal antibody to IFN-beta, while monoclonal antibody to IFN-gamma had no effect. However, monoclonal antibody to IFN-gamma only reduced the increased cytotoxic effect caused by rIFN-gamma by 25%. Catalase inhibited both basal monocyte cytotoxicity and the increase in cytotoxicity following addition of rIFN-gamma only slightly, suggesting that mechanisms other than the oxidative burst were active and could be induced by rIFN-gamma.     

Induction of tolerance to dinitrophenylated bovine IgG in pigs

Intraveneous administration of soluble 2,4-dinitrophenyl(DNP)-conjugated bovine IgG to pigs led to induction of tolerance to this antigen. A single dose of 100 mg soluble conjugate per animal resulted in complete tolerance which lasted at least 80 days. Lower doses led to partial tolerance. Induction of tolerance in pigs was shown to be a process taking several days to complete. Animals exhibiting tolerance when challenged by the homologous conjugate still synthesized significant amounts of antibody when challenged by the carrier protein or by a conjugate of the 2,4-dinitrophenyl group with a different carrier.