Molecular characterization and localization of human metabotropic glutamate receptor type 3.

A human brain cDNA library was screened using amplified human metabotropic glutamate receptor (mGluR) cDNA sequences as probes. The resulting clones included one containing the complete coding sequence of mGluR3. This sequence has 90% DNA sequence identity with rat mGluR3 and the predicted protein sequence has 97% identity. The mGluR3 cDNA was transfected in Chinese hamster ovary (CHO) cells. Stimulation of the expressed receptor by (2S,3S,4S)-alpha-(carboxycyclopropyl) glycine (L-CCG-I) resulted in a reduction of forskolin-stimulated cyclic AMP (cAMP) with EC50 values of 0.15-0.3 microM. A specific probe from the human mGluR3 clone was used to hybridise to Northern blots of mRNA from various human tissues and different brain regions. The mGluR3 mRNA is brain-specific, and is expressed in all the brain regions represented on the blot. In-situ hybridization studies on human brain sections confirmed this widespread distribution with expression in neurones in the cerebral cortex, caudate-putamen, thalamus and cerebellum.

Molecular characterization and localization of human metabotropic glutamate receptor type 4.

Oligonucleotides of consensus sequences from rat metabotropic glutamate receptor (mGluR) genes were synthesized and used to amplify human DNA by the polymerase chain reaction (PCR). Five unique human sequences homologous to these rat receptor genes were isolated including mGluR4. A human cerebellum cDNA library was screened using this amplified mGluR4 sequence as a probe and yielded clones which between them contained the complete coding sequence for human mGluR4. The coding sequence is very similar to the equivalent rat gene (90% DNA sequence identity and 97% predicted protein sequence identity). The mGluR4 cDNA was transfected in Chinese hamster ovary (CHO) cells and stable clonal cell lines were isolated. Stimulation of the expressed receptor by L-2-amino-4-phosphonobutyrate (L-AP4), L-glutamate or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) resulted in a reduction of forskolin-stimulated cyclic AMP (cAMP) with EC50 values of 0.2, 13 and 90 microM respectively. Quisqualate had little effect at concentrations up to 1 mM. In Northern blots mGluR4 mRNA appears to be brain-specific, and shows a distinct distribution (excluding the cerebellum), being expressed in the thalamus, hypothalamus and caudate nucleus. In situ hybridization studies on human brain sections confirmed this general pattern of distribution. The strongest mGluR4 mRNA signal was found in the cerebellar granule cells consistent with the reported distribution of mGluR4 in the rat brain. The major difference from the rat brain is the presence in the human brain of mGluR4 mRNA in the caudate nucleus and putamen.

The expression of mRNA for fibrinogen in megakaryocytes isolated from patients with T-cell lymphoma.

The expression of fibrinogen mRNA was studied by in situ hybridization in freshly isolated megakaryocytes in 14 newly-diagnosed patients: seven with non-Hodgkin's lymphoma (NHL), three with immune thrombocytopenic purpura (ITP) and four haematologically normal patients prior to coronary artery bypass surgery. Fibrinogen mRNA in megakaryocytes was not detected in ITP, B-cell lymphomas or in healthy donors. However, it was present in all patients with the high-grade T-cell lymphomas, both with and without thrombocytopenia.

Human colon cancer cell lines show a diverse pattern of nitric oxide synthase gene expression and nitric oxide generation.

A panel of human colonic adenocarcinoma cell lines was examined both for expression of mRNAs of the nitric oxide synthase (NOS) gene family and for evidence of enzymic activity based on citrulline and nitrite (NO2-) formation. Reverse transcription-polymerase chain reaction (RT-PCR), revealed that all lines (SW480, SW620, DLD-1 and WiDr) expressed mRNA for the Ca(2+)-dependent endothelial (e)NOS, while SW480 cells also expressed the Ca(2+)-dependent neuronal (n)NOS. The mRNA for the Ca(2+)-independent inducible (i)NOS was expressed both by cytokine-stimulated and by unstimulated SW480, SW620 and DLD-1 cells, but none was seen at any time in the WiDr cells. There was, however, little correlation between mRNA expression and enzymic activity based on citrulline and NO2- formation. Thus none of the cell lines exhibited measurable Ca(2+)-dependent NOS activity, while Ca(2+)-independent NOS activity was seen in all but the WiDr cells. Furthermore, DLD-1 cells generated citrulline with resultant NO2- formation only after stimulation with lipopolysaccharide (LPS) and/or cytokines, while SW480 and SW620 did so constitutively. Thus RT-PCR studies indicate that tumour cells of similar epithelial origin display a diverse pattern of NOS gene family expression, and parallel biochemical studies clearly indicate that such expression does not always result in measurable enzymic activity leading to the generation of NO.

The relationship between human megakaryocyte nuclear DNA content and gene expression.

Increased platelet reactivity has been implicated in various vascular diseases. Since the protein content of platelets is determined mainly by the megakaryocyte, alterations in megakaryocyte mRNA expression may influence platelet protein content and therefore activity. In order to determine whether DNA content (ploidy) of a megakaryocyte influences its mRNA expression, we have developed a method to investigate the relationship between megakaryocyte ploidy and gene expression. By measuring the ploidy and mRNA expression in individual cells, we have shown that in the physiological state there is an increase in mRNA expression for beta-actin, glycoprotein IIb and neuropeptide Y with increase in ploidy and that this increase levels off at high ploidy values. This may have relevance in the understanding of platelet reactivity in pathological events.

Constitutive and inducible nitric oxide synthases in human megakaryoblastic cells.

Human megakaryoblastic cells (Meg-01) were found to possess constitutive and express inducible nitric oxide (NO) synthase activities. The constitutive NO synthase was Ca+(+)- and NADPH-dependent, as is the NO synthase found previously in human platelets. Stimulation of Meg-01 cells by the cytokines interleukin-1 beta (0.15-32.5 ng/ml) and tumor necrosis factor-alpha (0.15-10 ng/ml) resulted in expression of the inducible, Ca+(+)-independent, NO synthase. This activity was increased by the addition of NADPH, tetrahydrobiopterin and sepiapterin as cofactors. Induction of this enzyme was accompanied by a decrease in the constitutive NO synthase activity, a phenomenon which was prevented or reversed by dexamethasone (1 microM). Thus, human early differentiated megakaryocytic cells can synthesize NO from L-arginine by both the constitutive and the inducible NO synthases. These findings indicate that these enzymes may play an important biological role in megakaryocyte and platelet functions.

Oxidation of low-density lipoprotein and macrophage derived foam cells.

Copper-oxidized LDL has many of the characteristics of the modified LDL generated in the artery wall during the initial stages of atherosclerosis. It is not, however, a chemically defined species but shows significant variations in both its chemical composition and behaviour in biological systems depending upon the extent to which the peroxidation reaction has occurred (Fig. 1). Taking care to define the extent of LDL modification we have used this form of oxidized LDL to investigate the effects on the macrophage of this potentially toxic particle. This cell, in contrast to endothelial cells, appears to be particularly well adapted to detoxify lipid peroxidation products since it possesses glutathione peroxidases capable of metabolizing oxidized LDL and responds to oxidized LDL by increasing its GSH content. Acetylated LDL had little or no effect on GSH levels showing that lipid loading per se or recognition by the macrophage scavenger receptor is not sufficient to induce the synthesis of this antioxidant. We have confirmed the observation that oxidized LDL does not activate expression of the gene for TNF and raise the possibility that PGE2 produced by the cells and possibly during the oxidation of LDL may be the mediator suppressing the synthesis of this cytokine. Our results support the hypothesis that the lipid-laden macrophage does not contribute to an inflammatory response in the artery wall and imply a protective role for the macrophage in scavenging oxidized LDL.

Identification of mRNA for PDGF B-chain in human megakaryocytes isolated using a novel immunomagnetic separation method.

A rapid and simple method has been developed for the separation of pure populations of intact human megakaryocytes from whole bone marrow. Megakaryocytes were specifically recognized using monoclonal antibodies coupled to magnetizable articles. Cells labelled with the magnetizable probe were separated from unlabelled cells by introduction of a magnetic field. The technique yields megakaryocyte suspensions with a purity of greater than 98%. Electron microscope examination showed that the ultrastructure of the isolated megakaryocytes was well preserved. Using this method of cell purification, we have investigated expression of the gene for platelet-derived growth factor (PDGF). RNA was isolated from cells harvested from either ribs or posterior iliac crest. The RNA was spotted onto nitrocellulose, and then hybridized using a c-sis riboprobe specific for PDGF B chain mRNA. We demonstrate that mRNA for PDGF B-chain is identifiable in samples of 50,000 cells. We conclude that PDGF is synthesized by the megakaryocyte.

Characterization of Leishmania major antigen-liposomes that protect BALB/c mice against cutaneous leishmaniasis.

Leishmania major antigen-liposomes prepared as dehydration-rehydration vesicles (DRV) and composed of equimolar amounts of L-alpha-distearoyl phosphatidylcholine and cholesterol confer high-level host-protective immunity against virulent homologous challenge to susceptible BALB/c mice. Physical and antigenic characterization of these protective liposomes is described. Both empty and L. major antigen-DRV were multilamellate and heterogeneous in size, ranging from 0.10 to 2.00 microns. Although the liposomes were made by using a crude mixture of promastigote antigens, lipophosphoglycan covered the liposome surface; this was demonstrated by immunogold electron microscopy. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed preferential entrapment of the 63-kilodalton promastigote protease (gp63) into the DRV. We suggest that our L. major antigen-DRV merit further study because of their preferential entrapment of these two host protective antigens together with their long in vivo half-life. In addition, this report illustrates that intravenous or subcutaneous immunization of BALB/c mice with the same limited subset of protective antigens, predominantly lipophosphoglycan and gp63, within DRV liposomes leads to either protection and low splenic interleukin-3 production or to nonprotection and high splenic interleukin-3 production, respectively. This was consistent with our hypothesis that differential antigen presentation after administration of the same immunogen by the intravenous or the subcutaneous route results in differential T-cell activation.

The relationship between megakaryocyte nuclear DNA content and gene expression.

Megakaryocytes are a distinct population of bone marrow cells that have the unique feature of increasing their DNA content without undergoing division. The biological effect of ploidy distribution on gene expression, receptor expression and protein synthesis is still unknown. Using molecular hybridization techniques, we have started a systematic analysis of mRNA expression in megakaryocytes for a number of proteins involved in clot formation. These data will be related to ploidy. Platelets are the unnucleated product of megakaryocytes, having their protein content derived from the precursor cell. Therefore, the understanding of the molecular mechanisms regulating megakaryocyte biology and the consequent type and reactivity of platelets produced is of fundamental importance in both physiological and pathological conditions.

Distinct IL-3 activation profile induced by intravenous versus subcutaneous routes of immunization.

Lymphoid cells from mice immunized i.v. or s.c. with Leishmania major antigens were analyzed for their capacity to produce lymphokines when stimulated with specific antigens in vitro. Spleen cells from BALB/c mice immunized by the s.c. route produced significantly higher levels of IL-3 and IL-3 mRNA than those from mice immunized by the i.v. route. The differential production of IL-3 was maintained at a wide range of antigen concentrations tested in vitro and for different culturing times. T cell enrichment procedures and treatment with CD4+ mAb in vitro confirmed the T cell nature of the IL-3 producer population. However, the IL-3 production in the two populations of spleen cells was equally high after Con A stimulation in vitro. The IL-2 production by the two populations of cells was also not significantly different after antigen or mitogen stimulation in vitro. To our knowledge, this is the first report of a differential synthesis of IL-3 mRNA and secretion of IL-3 induced by different routes of immunization followed by specific-antigen stimulation in vitro. These findings may also explain earlier observations that i.v. immunization with leishmanial antigen induces protection, whereas s.c. immunization leads to exacerbation of L. major infection, since IL-3 has been previously shown to promote leishmanial infection. The fact that the phenomenon also extends to other antigen systems suggests that this finding may have a broader implication in immune regulation and vaccine development.

Macrophage activation by interferon-gamma from host-protective T cells is inhibited by interleukin (IL)3 and IL4 produced by disease-promoting T cells in leishmaniasis.

BALB/c mice are highly susceptible to Leishmania major infection. They develop a progressive fatal disseminating disease even with a minimum infecting dose. However, these mice are able to contain the disease if they are exposed to sublethal gamma-irradiation shortly before infection. Earlier studies demonstrated that CD4+ T cells from mice which had recovered from infection (Tr) can adoptively transfer resistance. In contrast, CD4+ cells from mice with progressive disease (Ts) not only failed to protect, but can reverse the protective effect of the Tr cells. Spleen cells from BALB/c mice which had recovered from L. major infection or which had progressive disease were cultured with leishmanial antigens in vitro. The culture supernatant from spleen cells of recovered mice (TrSN) contains high levels of macrophage-activating factor (MAF) activity which can activate peritoneal macrophages to kill 51Cr-labeled P815 cells and to eliminate intracellular parasites as measured by the reduction in [3H]thymidine uptake by residual parasites released from macrophages following sodium dodecyl sulfate treatment. The MAF activity of TrSN parallels that of recombinant interferon-gamma (IFN-gamma). In contrast, culture supernatant of spleen cells from mice with progressive disease (TsSN) contains no detectable MAF but it is able to neutralize the MAF activity of TrSN. The MAF-inhibiting function of TsSN appears to be mediated by interleukin (IL)3 and IL4, since the MAF activity of TrSN and rIFN-gamma also can be inhibited by the addition of rIL3 and rIL4 but not by rIL1 or rIL2. Furthermore, the MAF-inhibiting activity of TsSN can be partially reversed by the addition of specific anti-IL3 or anti-IL4, but completely reversed by the combination of the two antibodies in vitro. These findings provide a mechanism for the immune regulation in leishmaniasis and a means by which the two subsets of CD4+ T cells influence each other through their modulation of macrophage function.

Vaccination against murine cutaneous leishmaniasis by using Leishmania major antigen/liposomes. Optimization and assessment of the requirement for intravenous immunization.

Efficacy of vaccination against cutaneous leishmaniasis in highly susceptible BALB/c mice was assessed comparatively by using radiation-attenuated promastigotes and colloidal Ag mixtures generated from a mixed Leishmania major (LV39) isolate (SLV39) and from a virulent cloned line (SVJ2) derived from the Jericho 2 L. major isolate. Dehydration-rehydration vesicle (DRV) liposomes were used as adjuvants. In optimization experiments phospholipid composition of DRV was varied, and the distearoyl derivative (DSPC) (liquid-crystalline phase transition temperature (Tc) 54 degrees C) of egg lecithin L-alpha-phosphatidylcholine was found to be superior to the dipalmitoyl derivative (DPPC, Tc 41.5 degrees C) and underivatized L-alpha-phosphatidylcholine (Tc -10 degrees C). The criteria studied were in vivo priming for a secondary in vitro proliferative response and the prepatency of lesion onset after L. major challenge of mice immunized once i.v. A single s.c. immunization with SLV39 either free or entrapped within DSPC liposomes primed spleen cells to produce significant levels of IL-3 when stimulated with SLV39 in vitro. In contrast, the i.v. route of immunization with the same Ag preparations led to little or no IL-3 production by the spleen cells. Despite development of significant T cell activation, both SLV39 and SVJ2 administered s.c., either free or entrapped within liposomes, were not protective. However, i.v. immunization four times with SVJ2 entrapped within DSPC liposomes induced a level of resistance comparable with that of 2 x 10(7) gamma-irradiated promastigotes in the stringent BALB/c L. major model. Although significant, protection conferred after i.v. immunization with SLV39/DSPC liposomes was less effective. These data therefore show that DSPC/DRV liposomes, although a good adjuvant for inducing protective immunity to cutaneous leishmaniasis, are not able to overcome the requirement for an i.v. route of immunization with the leishmanial Ag preparation. Additionally, they demonstrate a correlation between IL-3 secretion and non-protection. They also suggest that SVJ2 represents a better source of protective Ag than SLV39.

Effect of CD4 monoclonal antibody in vivo on lesion development, delayed-type hypersensitivity and interleukin 3 production in experimental murine cutaneous leishmaniasis.

Highly susceptible BALB/c mice subjected to adult thymectomy followed by prolonged (15 weeks), twice-weekly injections of a low dose (100 micrograms) of CD4 monoclonal antibody (MoAb) develop resistance to otherwise uniformly fatal and disseminating Leishmania major infection. In contrast, similar treatment with CD8 MoAb has no effect on the course of L. major infection. CD4 MoAb administered after the lesion establishment also has no effect. Mice which become resistant following CD4 MoAb treatment developed the classical delayed-type hypersensitivity (DTH) which persisted at 72 h after footpad injection with killed L. major promastigotes. A substantial degree of resistance can also be induced in the BALB/c mice with two i.v. high doses of 500 micrograms of CD4 MoAb given immediately prior to L. major infection. The treated mice developed classical DTH and significantly smaller lesions. The spleen cells from these mice also produced significantly lower levels of IL-3 compared to that of untreated control mice when cultured with L. major antigens in vitro. In contrast, genetically resistant CBA mice treated with CD4 MoAb developed significantly larger lesions but lower levels of classical DTH compared to untreated mice. These data confirmed and extended earlier reports on the prophylactic effect of CD4 MoAb in susceptible BALB/c mice and the disease exacerbative effect in the resistant CBA mice. The data also further illustrate the direct correlation between classical DTH and resistance, and the inverse correlation between IL-3 production and resistance in experimental cutaneous leishmaniasis.

Susceptibility to murine cutaneous leishmaniasis correlates with the capacity to generate interleukin 3 in response to leishmania antigen in vitro.

Spleen cells from genetically susceptible BALB/c mice infected with Leishmania major produced higher levels of IL-3 in response to leishmania antigens or concanavalin-A in vitro compared to that of genetically resistant CBA mice throughout the course of infection. The capacity to generate IL-3 in BALB/c mice increased with disease progression. The correlation between susceptibility to L. major infection and the capacity of spleen cells to produce IL-3 also extends to other mouse strains. Thus genetically highly resistant CBA and Biozzi Low mice are low IL-3 producers, whereas the highly susceptible BALB/c and Biozzi High mice are high IL-3 producers. The resistant C57BL/10 and C3H mice produce intermediate levels of IL-3. BALB/c mice recovered from L. major infection following a sublethal dose of gamma-irradiation are refractory to further infection. The capacity of the spleen cells from these cured mice to produce IL-3 upon a challenge infection was similar to those of the resistant CBA mice. The IL-3 generated by activated T cells was measured by IL-3 dependent cell lines, 32D and FDC-P2. The spleen cells from infected BALB/c mice also contain a population of IL-3 responding cells whose number increases as disease progresses. A similar population of IL-3 responding cells was barely detectable in the resistant CBA mice or BALB/c mice rendered immune by prior gamma-irradiation. These results therefore demonstrate a direct correlation between the susceptibility to L. major infection and the capacity of splenic T cells from infected mice to produce a continuous elevated level of IL-3. The possible role of IL-3 in the immune regulation of cutaneous leishmaniasis is discussed.

Prophylactic immunization against experimental leishmaniasis. VI. Comparison of protective and disease-promoting T cells.

In previous studies, we reported that mice immunized i.v. with lethally irradiated Leishmania major promastigotes developed substantial resistance to a subsequent L. major infection. However, such protection could be totally suppressed by prior s.c. injection with the same antigens. Both the protective immunity and the inhibition of its induction could be adoptively transferred with specific Lyt-2- T cells. Here, we present evidence showing that protection and disease promotion resulting from i.v. or s.c. immunization, respectively, are mediated by functionally distinct subsets of T cells. In a series of titration experiments, it was found that freshly isolated T cells derived from prophylactically i.v. immunized BALB/c mice were either protective (greater than 10(7) cells/recipient) or ineffective (less than 10(7) cells/recipient). No exacerbation of disease was observed at any dose. Conversely, T cells from mice immunized s.c. either accelerated disease development and inhibited protective immunization (greater than 10(7) cells/recipient) or had no effect (less than 10(7) cells/recipient). No protection was observed at any dose tested. In mixed transfer experiments, increasing numbers of T cells from s.c. immunized donors progressively inhibited the protective effect of T cells from i.v. immunized donors. Supernatant of T cell cultures from protectively immunized donors contained substantial macrophage-activating factor whereas such activity was not detectable in the supernatant of T cell culture from s.c. immunized donors. Analysis by flow cytometry showed that the spleen and lymph nodes of normal, i.v., or s.c. immunized BALB/c mice contained similar ratios of L3T4+ cells and Lyt-2+ cells.

Is there an interleukin 2 inhibitor in human serum?

Normal mouse serum has been shown to contain an inhibitor of interleukin 2 (IL-2). Here we report that a molecule with similar activity cannot be found in normal human serum (NHS). Although NHS inhibited the IL-2-dependent proliferation of mouse CTLL cells, as expected of an IL-2 inhibitor, it also had inhibitory activity on IL-3-dependent cells and was cytolytic to IL-2-independent mouse cells as measured by a 51Cr release assay, indicating a nonspecific effect. In addition, NHS had no effect on the IL-2-dependent proliferation of human peripheral blood T-cell blasts. Fractionation of NHS by size exclusion HPLC failed to separate cytolytic activity from any putative true IL-2 inhibitor activity. The cytolytic component was not related to immunoglobulin since it had a molecular weight of 50,000 to 60,000 and was not bound by protein-A-Sepharose. However, its molecular weight, heat lability, and trypsin sensitivity suggest it to be a protein.

Suppression of interleukin-2 production by macrophages in genetically susceptible mice infected with Leishmania major.

Spleen cells from BALB/c mice infected with 2 X 10(7) L. major promastigotes and developing progressive disease produced significantly lower levels of interleukin-2 (IL-2) in response to concanavalin A stimulation than did spleen cells from uninfected mice. In contrast, spleen cells from sublethally irradiated and infected mice, which were able to contain lesion development, produced significantly higher levels of IL-2. The increase in IL-2 production closely paralleled lesion regression. Mice protectively immunized by four intravenous injections with lethally irradiated promastigotes also produced enhanced levels of IL-2, which were sustained after challenge infection. In contrast, spleen cells from BALB/c mice given four s.c. injections of irradiated promastigotes produced high levels of IL-2 before but not after infection. These mice eventually produced levels of IL-2 indistinguishable from those of unimmunized mice with progressive disease. There is thus an inverse relation between disease progression and the ability of spleen cells to produce IL-2. Spleen cells from mice with uncontrolled disease not only produced lower levels of IL-2 but also impaired IL-2 production by normal spleen cells. The ability to inhibit IL-2 was abrogated by passing the cells through a Sephadex G-10 column, removal of plastic adherent cells, and removal of carbonyl iron-ingesting cells. Furthermore, Sephadex G-10 column-treated and plastic adherent, nonspecific esterase-positive spleen cells from mice with progressive disease were able to suppress IL-2 production by normal splenic T cells. The suppressive activity of the adherent cells was not affected by treatment with anti-Thy-1.2 antibody and complement. In contrast, adherent spleen cells from uninfected mice were devoid of such suppressor activity. The depressed IL-2 production by spleen cells from progressively infected mice could be restored to that of normal spleen cells by the addition of indomethacin to the culture. There was however, no correlation between IL-2 production and IL-1 activity in infected or immunized BALB/c mice. Thus, it appears that the suppression of IL-2 production is mediated by prostaglandins elaborated by macrophages from chronically infected mice.

Serum IL-2 inhibitor in mice. I. Increase during infection.

Serum from normal mice contains an inhibitor of interleukin-2 (IL-2) which probably interacts directly with IL-2. Athymic mice and normal mice kept under specific pathogen-free conditions do not show this activity, whereas mice infected with malaria parasites have increased serum levels of inhibitor. This IL-2 inhibitor may play an important part in regulating T-cell function.

Serum IL-2 inhibitor in mice. II. Molecular characteristics.

The molecular characteristics of a serum IL-2 inhibitor were determined by fractionating active sera from normal and malaria-infected mice, and assaying inhibitory activity in blocking production and expression of IL-2 activity. Using isoelectric focusing and ion exchange chromatography, the activity resolved in a single peak (pI = 6.2). HPLC gel filtration resolved two peaks of activity (50,000 and 25,000 MW) which are probably related. The molecule is precipitated by 50% saturated ammonium sulphate, and is destroyed by heating above 56 degrees or by acidification below pH 4.