Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac-2/epsilon BP) of the S-type lectin family: role in IgE-dependent activation.

It has been suggested that neutrophils may be involved in the late-phase reaction of immunoglobulin E (IgE)-dependent hypersensitivity states. However, the identity of neutrophil-associated molecules inducing the release of mediators remains unclear. In this report, we demonstrate that human neutrophils from normal donors or from patients with inflammatory disorders could bind myeloma IgE proteins, especially after desialylation. Northern blot, immunoprecipitation, and flow cytometry analyses revealed that neutrophils did not express Fc epsilon RII/CD23, but rather Mac-2/epsilon binding protein (BP), belonging to the S-type lectin family. Similarly to IgA used as positive control, myeloma IgE proteins, as well as polyclonal IgE antibodies with or without antibody specificity, were both capable of inducing a neutrophil respiratory burst. Anti-Mac-2 but not anti-CD23 mAb strongly decreased the IgE-dependent activation of neutrophils, induced either by the specific antigen or by anti-IgE antibodies. These findings open new perspectives on the functional role of neutrophils in IgE-associated diseases including allergic states or parasitic infections.

Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages.

An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE-dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2).

Evidence of non-deficient low-density lipoprotein receptor patients in a pool of subjects with clinical familial hypercholesterolemia profile.

For this study, we selected 41 adult patients with the classic clinical diagnosis of heterozygous familial hypercholesterolemia (FH), which is characterized by a low-density lipoprotein (LDL) cholesterol level above the 95th percentile, xanthomas, and/or personal or familial cardiovascular history. We used an indirect immunocytofluorimetric assay to classify these 41 subjects according to LDL receptor function on lymphocytes. We found that LDL receptor activity was normal in nine patients. A large study of plasma lipid, lipoprotein, and apolipoprotein levels found no significant difference between patients with and without LDL receptor defect. Familial defective apolipoprotein (apo) B-100 (FDB) and LDL-binding defects were not found in the nine patients without LDL receptor defect. These results suggest that other defects in the regulation of lipoprotein metabolism are capable of giving rise to a clinical and biochemical disorder indistinguishable from classic FH.

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.

[Infection of T lymphocytes by Trypanosoma cruzi. Possible role of CD3 and HLA-DR]. / Infection des lymphocytes T par Trypanosoma cruzi. Rôle possible du CD3 et du HLA-DR.

The mechanisms by which the causative agent of Chagas' disease impair its host's immune response are of paramount importance but poorly understood. Results presented in this paper show for the first time that Trypanosoma cruzi trypomastigotes infect T lymphocytes in vitro and more interestingly in vivo, and that trypomastigotes released from infected cells are infectious. In addition treatment of purified human T lymphocytes with McAb against CD3 and HLA-DR antigens significantly inhibited parasite infection. T. cruzi antigens were detected on the membrane of infected T cells and could therefore represents targets for cytotoxic mechanisms. These results might have important consequences for the understanding of the dramatic disruption of immune response observed during Chagas' disease and more generally provide additional information on T lymphocyte infection by pathogens.

Selectivity of mediators released by eosinophils.

The presence of receptors for IgE on eosinophils has drawn the attention on their direct participation in IgE-dependent hypersensitivity reactions. Surface IgE antibodies were detected on eosinophils from allergic patients. The addition of the specific allergen or anti-IgE antibodies to such purified eosinophils induced the release of eosinophil peroxidase, but not of eosinophil cationic protein. These findings associated with results obtained by using electron microscopy and immunogold staining of the various antibodies directed against the granule proteins allowed us to suggest a selectivity in the mediators released by eosinophils. In addition, preliminary results concerning the existence and the functional role of a receptor for IgA on eosinophils are reported, leading to the concept of a particular interaction of eosinophils with immunoglobulins present in the tissues and their participation in local immune responses.

Leishmania chagasi: in vitro differentiation of promastigotes monitored by flow cytometry.

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The "forward scatter" intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/micron 2) and in exoplasmic fracture faces (about 1000/micron 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freeze-fracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro cultures.

Infectivity of Leishmania braziliensis promastigotes is dependent on the increasing expression of a 65,000-dalton surface antigen.

Sequential development of Leishmania braziliensis promastigotes from a noninfective to an infective stage was demonstrated. The generation of infective forms was related to their growth cycle and restricted to stationary stage organisms. Using immunofluorescence techniques, we have noticed that the binding of a monoclonal antibody (mAb) against L. braziliensis (VD5/25) increased progressively as the promastigotes developed in culture and was maximal with the infective forms. This antigenic differentiation was not detected with an anti-L. braziliensis polyclonal rabbit antiserum, suggesting that only a few epitopes, including that recognized by VD5/25, have their expression effectively increased on the surface of infective promastigotes. Immunoprecipitation of lysates of surface-iodinated L. braziliensis promastigotes with this mAb revealed two proteins of apparent 65,000 and 50,000 Mr, the 50,000 Mr protein probably representing the unreduced form of the major surface glycoprotein described in several species of Leishmania (GP65). The increasing expression of this epitope was not found with L. chagasi promastigotes, but seems to occur with the parasites from the L. mexicana complex. Intracellular survival of L. braziliensis was completely inhibited when the infective promastigotes were treated with VD5/25. It appears, therefore, that the increasing expression of GP65 on the promastigote surface represents an essential mechanism of leishmania survival in the macrophage.

Role of nitric oxide in the regulation of lymphocyte apoptosis and HIV-1 replication.

Nitric oxide (NO) has been implicated in certain immunopathogenetic mechanisms during the course of infection with human immunodeficiency virus (HIV). We have evaluated the levels of NO release and lymphocyte apoptosis in peripheral blood mononuclear cell (PBMC) cultures from HIV-1 infected subjects and healthy controls. We have also examined these 2 parameters in parallel cultures maintained under conditions where either NO synthesis was inhibited or high level of NO was present. Nitrite contents in culture supernatants were measured as the stable end products of the released NO. Levels of spontaneous apoptosis and activation-induced cell death (AICD) by anti-CD3 or by phytohemagglutinin were evaluated using flow cytometry. Additional experiments were also aimed at addressing a potential link between NO synthesis and HIV-1 replication in human monocyte-derived macrophages (MDMs). Acutely infected MDMs with HIV-1Bal were maintained in culture, without any additional activation signal, for a period of 14 days. Nitrites in the supernatants and mRNA accumulation of the inducible NO synthase (iNOS) in infected cells were assessed over the whole culture period. In addition, the effect of blocking NO synthesis during and after infection of MDMs, using an inhibitor of NO, was evaluated on the level of viral replication as measured by the presence of P24 antigen in the supernatants. Similarly, the effect on HIV replication of high NO levels in MDM cultures, supplied by a donor of NO during the 24 h period of infection, was also studied. We conclude that no elevation in NO release could be detected in PBMC cultures from HIV-1 infected subjects and that modulation of NO content may slightly regulate the level of spontaneous lymphocyte apoptosis but not that of AICD. Infection of MDMs with HIV-1 does not seem to induce detectable NO release or iNOS mRNA accumulation. Similarly, neither inhibition of NO synthesis nor the presence of high NO levels during the infection period could modify the outcome of virus replication in macrophages.

[Presence and functional role of an epitope of the second receptor for IgE (FcE RII) in mast cells and basophils in the rat]. / Présence et rôle fonctionnel d'un épitope du second récepteur pour l'IgE (FcE RII) sur les mastocytes et les basophiles de rat.

Mast cells and basophils express the high affinity IgE receptor (FcERI) whereas the low affinity receptor for monomeric IgE (FcE RII) is present on macrophages, lymphocytes, eosinophils, platelets and Langerhans cells. Recent studies confirmed that the two receptors were totally distinct. The present work shows that a monoclonal antibody (BB10), able to bind to FcE RII on different cell populations, interacts with FcE RI expressing cells: rat peritoneal mast cells and a rat basophilic leukemia cell line (RBL 2 H 3). The structure recognized by BB10 is distinct from FcE RI and modulates the IgE-dependent histamine release. In conclusion, it appears that a common epitope with FcE RII is present on mast cells and basophils and that a functional relation might exist between this structure and FcE RI.

A suppressive lymphokine of platelet cytotoxic functions.

The in vitro stimulation of mononuclear cells from human peripheral blood with mitogens is known to induce the release of factors (monokines and lymphokines) that possess distinct biologic activities. The present data describe the presence in Con A- and antigen-stimulated T cell supernatants (of man or rat) of a factor able to inhibit, in a dose-dependent manner, the platelet cytotoxicity toward the young larvae of Schistosoma mansoni. The production of oxygen metabolites by IgE-coated platelets, stimulated by anti-IgE or the specific antigen, was, likewise, strongly inhibited by this lymphokine. The producing T lymphocyte subpopulation was identified as OKT 8+. This suppressive lymphokine of platelet functions had an m.w. of 15,000 to 20,000 and a pI of 4.6. It was heat- and acid-stable and sensitive to trypsin and proteinase K, but neuraminidase had no effect on its activity. This platelet suppressive activity was specifically absorbed by platelet membrane, suggesting its action through the binding to a receptor.

Trypanosoma cruzi: infection of T lymphocytes and their destruction by antibody-dependent cell-mediated cytotoxicity.

We have demonstrated, with optical and transmission electron microscopy, that Trypanosoma cruzi trypomastigotes infect and multiply inside T lymphocytes. The infection rate we have observed in T cells was similar to that seen in the case of macrophage or polymorphonuclear cell infection. Flow cytofluorometric analysis of T lymphocytes purified from mice in the acute phase of the disease, revealed the presence of parasite-derived antigens on their surface. These antigens appear to be specific to T. cruzi and they could be the result of intracellular parasite antigens as well as adsorption of T. cruzi antigens on the surface of noninfected T cells. Antibodies recognizing these surface antigens were present in both T. cruzi-infected mouse and human sera. They were able to induce antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of nonimmune mononuclear cells both in autologous and in heterologous combinations. Consequently, we provided evidence suggesting that T lymphocytes could be destroyed during the acute phase of Chagas' disease either by cell infection or by an ADCC mechanism against cells bearing parasite antigens on their surface. Thus, the ability of trypomastigotes to invade T cells may play a crucial role in the immunopathogenesis characteristic of Chagas' disease.

[Participation of the IgE receptor in the toxicity of blood platelets against schistosomes]. / Participation du récepteur pour l'IgE à la toxicité des plaquettes sanguines contre les schistosomes.

Human blood platelets, incubated with the serum from patients with schistosomiasis or allergic asthma, became cytotoxic for schistosome larvae, in an allergen-specific mechanism linked to surface bound IgE on platelets. Cytotoxic factors, among which oxygen metabolites might be involved, were able to kill schistosomula through platelet-retaining filters. Flow cytometry showed that platelets bearing IgE receptors represented a subpopulation, the percentage of which significantly increased in patients with high levels of circulating IgE.

Human eosinophils express CR1 and CR3 complement receptors for cleavage fragments of C3.

The functional and antigenic characteristics of C3 receptors expressed on human eosinophils were investigated using rosette assays with sheep erythrocytes coated with C3 fragments and flow cytometric analysis of cells stained with anti-receptor antibodies. Purified peripheral blood eosinophils from 13 patients with hypereosinophilia expressed CR1 antigens. In 8 patients, a mean of 14 + 9.5% eosinophils formed C3b-dependent rosettes that were inhibited by F(ab')2 anti-CR1 antibodies. This number increased to 33% following stimulation with leukotriene B4 (LTB4) (10(-7) M). Similar numbers of C3b rosettes were formed by hypodense and normodense eosinophils. Eosinophils from 2 patients from this group expressed 20,000 125I-labeled monoclonal anti-CR1 antibody binding sites/cell. In another group of patients, 55 +/- 9% eosinophils spontaneously formed C3b-dependent rosettes that could not be enhanced by LTB4. In all patients, a mean of 16 +/- 9% eosinophils formed cation-dependent rosettes with C3bi-bearing intermediates that were inhibited by anti-CR3 antibody OKM1. All eosinophils stained with monoclonal antibodies against the alpha chain of CR3. There was no C3d-dependent rosette formation with eosinophils and no eosinophils stained with monoclonal anti-CR2 antibody. Thus, human eosinophils express CR1 and CR3. Since CR3 is required for the adhesion of granulocytes to surfaces and antibody-dependent cellular cytotoxicity of neutrophils, the interaction of C3 fragments with CR3 and CR1 on eosinophils may be of importance in eosinophil-mediated damage of opsonized targets.

Tissue expression of the Schistosoma mansoni 28 kDa glutathione S-transferase.

The expression of the Schistosoma mansoni 28 kDa glutathione S-transferase (Sm28) was studied using molecular (PCR, in situ hybridization), and immunocytochemical techniques. The presence of Sm28 was demonstrated in all developmental stages of the parasite except the intra-uterine immature egg. In the parenchyma of male and female adult worms the distribution of Sm28 was limited to a subpopulation of parenchymal cells and to the dorsal tubercles of the male. The tegument, the muscles, the digestive tract, the neural mass, the vitelline glands, and mature gametes were not immunoreactive. Immature germinal cells in both sexes, and the ootype in the female genital system, were found to express Sm28. Deposits of immunoreactive material on host skin following cercarial penetration, exfoliation from the male tubercles, and especially emission of Sm28 from eggs in hepatic granulomas are suspected to be a source of antigen during the parasite infection. The reduction in worm fecundity previously observed in immunization experiments may result from an antibody response directed against Sm28 present in the ootype. There was no cross-reactivity observed, under the experimental conditions used, between the anti-Sm28 sera and either vertebrate or invertebrate host tissue.

Development of metacyclic Leishmania promastigotes is associated with the increasing expression of GP65, the major surface antigen.

Using immunofluorescence techniques and flow microfluorometry analysis, we have demonstrated that the binding of a monoclonal antibody (VD5/25) produced against GP65, the major surface antigen of Leishmania braziliensis, increased on the surface of stationary-phase promastigotes from all the New World Leishmania species causing mucocutaneous or cutaneous disease as compared with the log-phase parasites. In addition, a sequential development of Leishmania amazonensis promastigotes from a non-infective to an infective stage was demonstrated. Indeed, promastigotes in the stationary phase (days 6-7) were found to be far more infective than those in the logarithmic phase of growth (day 3) both in vitro for mouse peritoneal macrophages and in vivo for BALB/c mice. The intracellular survival and multiplication of L. amazonensis were significantly inhibited when infective promastigotes were treated with the VD5/25 monoclonal antibody. The increasing expression of GP65 on the promastigote surface may thus contribute to Leishmania infectivity. This seems to represent a characteristic mechanism applicable to all New World Leishmania species studied.

Recombinant human interferon-gamma induces increased IgE receptor expression on human platelets.

Human recombinant interferon-gamma (IFN-gamma) significantly increased the expression of receptors for IgE (Fc epsilon RII) on blood platelets. Fc epsilon RII was measured by specific binding of 125I-labeled IgE or flow cytometry experiments. Scatchard analysis of 125I-labeled IgE binding curves revealed that treatment with IFN-gamma increased the number of Fc epsilon RII but did not change the value of the association constant of Fc epsilon RII for 125I-labeled IgE. IFN-alpha had no effect on the expression or affinity of Fc epsilon RII. In addition to Fc epsilon RII, IFN-gamma also modified the expression of the glycoprotein IIb-IIIa complex on the platelet membrane.

Schistosoma mansoni-specific rat T cell clones. II. Different effects of adult worm-specific T cell clones in immunocompetent and nude infected rats.

The in vivo functional activities of two highly proliferating helper rat T cell clones (E23 and G5) specific for the excretory-secretory antigens of Schistosoma mansoni adult worms were investigated. When injected into infected immunocompetent rats, both clones increased the antibody response against the 30-40-kDa schistosomulum surface antigens, but failed to induce an immune protection. In contrast, when the same clones were injected into infected nude rats, a high degree of protection was obtained. In this latter case the absence of detectable specific antibody response, whether of IgE or IgG isotype, suggested that parasites were destroyed by an antibody-independent mechanism, i.e. macrophages activation by lymphokines. Indeed supernatants obtained from T cell clones specifically restimulated with schistosome antigens expressed a macrophage activated activity similar to interferon-gamma. Following incubation with these supernatants or with the active fractions, macrophages exhibited a significant schistosomulicidal activity and both clones were shown to transfer an antigen-specific delayed-type hypersensitivity reaction to normal rats. Taken together these results demonstrate that, depending on the immune status of the host, antigen-specific T cell clones can function differently and consequently that one function associated with one type of lymphokine could be favored.

Cytokine analysis at the single cell level and lymphoproliferative responses to mycobacterial antigens in HIV-1 patients with successful virologic response to potent antiretrovirals.

Immunologic parameters, known to be grossly abnormal in HIV-1-infected subjects, were analyzed in 22 patients with sustained viral load suppression (<200 copies/ml) following long-term highly active antiretroviral therapy (HAART). Responses were compared with those from 18 HIV-seronegative healthy controls. Persistent phenotypic alterations in patients' blood mononuclear cells were minimal, though the percentages of lymphocytes that could be activated to produce interleukin-2 (IL-2) remained severely depressed. Using lymphoproliferative assays, a striking deficit in the capacity of patients to respond to the common mycobacterial antigens and particularly to recombinant heat-shock proteins paralleled the absence of responses to virus p24 antigen. In view of the important immunoregulatory role of stress proteins, these findings reveal profound functional deficiencies and persistent immune dysregulation in HIV-1 patients, despite successful HAART and a considerable recovery of CD4+ lymphocyte numbers. Rational immunotherapeutic approaches should be aimed to correct the characterized immune abnormalities.