Determination of sensitivity of fresh leukemia cells to immunotoxins.

In clinical practice, sensitivity of malignant cells to a given immunotoxin remains hypothetical, since standard test systems such as the protein synthesis inhibition assay or the cloning assay are not appropriate. This study evaluated the feasibility of a semi-routine procedure based on dye exclusion assay enumerating the percentage of living cells after fluorescein diacetate-propidium iodide staining. The validity of the method was evaluated using five different subclones derived from the CEM cell line, which expressed a wide range of sensitivity to T101 A-chain immunotoxin. The comparison between dye exclusion assay and standard test systems suggested that this method might allow an easy and reproducible semi-quantitative evaluation of the sensitivity of leukemia cells. In a series of 21 patients suffering from various blood diseases in which the malignant cells expressed the T65 antigen, dye exclusion assay could detect clear T101 immunotoxin cell sensitivity in about 50% of the cases. The mean density of T65 antigen on malignant cells was found to influence dramatically the sensitivity of target cells to T101 immunotoxin.

Immunopharmacological profile of SR 31747: in vitro and in vivo studies on humoral and cellular responses.

In our preceding paper, we demonstrated that both human and rat lymphocytes possess saturable high-affinity binding sites for the new sigma ligand SR 31747. Here we investigate the potential activity of this ligand on immune responses. In vitro, our study shows that SR 31747 exerts a concentration- and time-dependent inhibition of proliferative response to mitogens on mouse and human lymphocytes without affecting cell viability. This suppressive effect elicited by SR 31747 occurs over a concentration range which correlates with the pharmacological profile of the molecule in binding assays, strongly suggesting that SR 31747 acts through a receptor-mediated process. We showed that the SR 31747 effect, which was observed on purified T lymphocytes, affects a late event in the activation process which occurs after the G1 during the S phase of the cell cycle. Interestingly, no anti-proliferative effect was observed in a variety of tumor cell lines, supporting a specific effect limited to normal immune cells. In vivo, in mice, treatment with SR 31747 prevented both graft-versus-host disease and delayed-type hypersensitivity granuloma formation, while antibody response to sheep red blood cells was not affected. These results strongly suggest that the sigma-related receptor recognized by SR 31747 is very likely coupled to a biological function of lymphocytes.

Allosteric modulation of peripheral sigma binding sites by a new selective ligand: SR 31747.

The interactions of a new compound SR 31747 with sigma sites were examined in rat spleen membranes and in human peripheral blood leukocytes (PBL). Nanomolar concentrations of SR 31747 selectively inhibited in a non-competitive manner the binding of the prototypic sigma ligands [3H](+)-pentazocine, [3H](+)-3PPP and [3H]DTG on rat spleen membranes. Characterization of SR 31747 binding sites using [3H]SR 31747 as a ligand showed that this compound binds reversibly, with high affinity to one class of sites on rat spleen membranes (Kd 0.66 nM, Bmax 5646 fmol/mg protein). The pharmacological profile of [3H]SR 31747 binding sites was consistent with the presence of specific sites distinct from classical sigma 1 and sigma 2 receptor subtypes strongly suggesting an allosteric modulation of sigma sites by SR 31747. Similarly, [3H]SR 31747 binding sites were demonstrated on human PBL and also on purified subpopulations of human mononuclear cells (granulocytes, NK cells, T4, T8 and B lymphocytes). Administered to mice by i.p. or oral route 30 min before sacrifice, SR 31747 strongly inhibited the binding of [3H](+)-3PPP to mice spleen membranes with ED50 values of 0.18 and 1.43 mg/kg, respectively. Taken together these results could suggest a potential immunological activity of SR 31747 either directly or through allosteric modulation of peripheral sigma sites.

Peripheral benzodiazepine receptor modulation with phagocyte differentiation.

Peripheral benzodiazepine receptor (PBR) was found to be less expressed in the immature phagocytic HL-60 and U-937 cell lines than in the more mature monocytic THP-1 cell line. Cell differentiation by several agents induced a strong enhancement of PBR density on these three phagocytic cell lines but not on the lymphocytic CEM cell line. Detailed analysis of phorbol 12-myristate 13-acetate-treated THP-1 cells showed an increased PBR expression and the rise came along with an increase of CD11a and CD11b antigens and a secretion of macrophagic cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-8. Quantitation of mRNA using polymerase chain reaction (PCR)-based technique showed that overexpression of PBR did not parallel mRNA expression, indicating a gene-independent regulation. These results suggest that PBR predominance on phagocytic cells could be related to maturation process.

Distribution profile and properties of peripheral-type benzodiazepine receptors on human hemopoietic cells.

The cellular localization of peripheral-type benzodiazepine receptors (PBRs) was characterized in several human blood cell subpopulations including erythrocytes, platelets, monocytes and polymorphonuclear neutrophils (PMN), B, NK, T8 and T4-cells. Pharmacological properties of the PBR were established by binding studies and PBR mRNA expression was measured by quantitative polymerase chain reaction based method. These data clearly indicate 1) the PBR is pharmacologically homogeneous in the various types of blood cells, 2) the rank order of PBR cell density is monocytes = PMN > lymphocytes >> platelets > erythrocytes, 3) the PBR appears to be transcriptionally regulated since mRNA levels are roughly correlated with PBR density.

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Development of a monoclonal antibody to immuno-cytochemical analysis of the cellular localization of the peripheral benzodiazepine receptor.

Based on the amino acid sequence deduced from the cloned human peripheral benzodiazepine receptor (PBR) gene, monoclonal antibody (Mab 8D7) was produced against the C-terminal fragment of the receptor. Immunoblot experiments, performed against purified PBR, indicated that the antipeptide antibody recognized, under denaturing conditions, the corresponding amino acid sequence of the PBR. When mitochondrial membranes from PBR transfected yeast or from THP1 and U937 cells were used on immunoblot analysis, a high level of immunoreactivity was observed at 18 kDa, the PBR molecular mass deduced from cDNA, establishing the specificity of the antibody for the receptor. Moreover, binding experiments realized with intact mitochondria demonstrated that the immunogenic sequence was accessible to the antibody indicating that the C-terminal fragment of the PBR faces the cytosol. Using this Mab we developed a technique which allowed precise quantification of PBR density per cell. Furthermore, cellular localization studies by flow cytometric analysis and confocal microscopy on cell lines displaying different levels of PBR showed that Mab 8D7 was entirely colocalized with an antimitochondria Mab.

Involvement of peripheral benzodiazepine receptors in the protection of hematopoietic cells against oxygen radical damage.

Several putative functions have been attributed to the peripheral benzodiazepine receptor (PBR), but its precise physiologic role has not been elucidated. In the present study, we investigated PBR function by quantifying this receptor in leukocyte subsets from healthy donors and in leukemic blasts from lymphoid and myeloid lineages. Using a monoclonal antibody (MoAb) directed against the human PBR and a quantitative flow cytometric assay, we found that phagocytic cells from healthy donors displayed a higher level of PBRs than lymphocytes or natural killer (NK) cells. Among the lymphoid lineage, thymocytes and IgD-negative B cells expressed the lowest levels. However, because of the wide heterogeneity of PBR levels among 42 acute or chronic lymphoid and myeloid leukemias, it was not possible to assign PBR expression to a stage of maturation or a cell lineage. Although the PBR displayed a mitochondrial subcellular localization, its expression was not correlated with the mitochondrial content, suggesting a modulation of PBR density at the level of the mitochondria. This modulation was confirmed when we studied in detail the PBR expression during T-cell development by both flow cytometry and confocal microscopy. We found that the PBR was expressed with a bimodal profile during T-cell development, identical to the one observed with the proto-oncogene, Bcl-2. The high similarity in the expression of both the PBR and the Bcl-2 proto-oncogene in T-cell and B-cell subsets, their common mitochondrial localization, and the observation of high quantities of PBR in phagocytic cells, which are known to produce high levels of radical oxygen species, suggested that PBRs may participate in an antioxidant pathway. Indeed, a strong correlation was established between the ability of hematopoietic cell lines to resist H202 cytotoxicity and their level of PBR expression. Demonstration of the role of PBR in the protection against H202 was obtained by transfecting JURKAT cells with the human PBR cDNA. Transfected cells exhibited increased resistance to H202 compared with wild-type cells, suggesting that PBR may prevent mitochondria from radical damages and thereby modulate apoptosis in the hematopoietic system.

Optimal elimination of leukemic T cells from human bone marrow with T101-ricin A-chain immunotoxin.

In view of bone marrow purging before autologous transplantation in T cell malignancies, an anti-human T cell immunotoxin (IT) has been prepared by coupling ricin A-chain to the monoclonal antibody T101 that binds the T1 differentiation antigen expressed by T lymphocytes as well as by T cell-derived hematologic malignancies. Using a sensitive and reliable clonogenic assay, optimal conditions were defined for the elimination of clonogenic human T leukemic cells among bone marrow cells. Maximal cytoreduction was obtained with IT at a dose of 2 micrograms/mL in the presence of 10 mmol/L NH4Cl. This treatment led to the reduction of more than six orders of magnitude of T101-positive clonogenic leukemic cells, with no harm to T101-negative cells. Moreover, we observed no toxicity of IT to human hematopoietic stem cells (CFU-GEMMT) derived from bone marrow of healthy volunteers. Thus, pretreatment of bone marrow samples with IT plus NH4Cl offers a safe, simple, reliable, and highly efficient means to eliminate undesirable leukemic T cells from the graft.

Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1.

The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

Signaling pathway associated with stimulation of CB2 peripheral cannabinoid receptor. Involvement of both mitogen-activated protein kinase and induction of Krox-24 expression.

Cannabinoids, known for their psychoactive effects, also possess immunomodulatory properties. The recent isolation and cloning of the G-protein-coupled peripheral cannabinoid receptor (CB2), mainly expressed in immune tissues, have provided molecular tools to determine how cannabinoid compounds may mediate immunomodulation. We here investigated the CB2 signaling properties using stably transfected Chinese hamster ovary cells expressing human CB2. First, we showed that stimulation by a cannabinoid agonist activated mitogen-activated protein (MAP) kinase in time- and dose-dependent manners. The rank order of potency for MAP kinase activation of cannabinoid agonists correlated well with their binding capacities. Second, we demonstrated that, following MAP kinase activation, cannabinoids induced the expression of the growth-related gene Krox-24, also known as NGFI-A, zif/268, and egr-1. Pertussis toxin completely prevented both MAP kinase activation and Krox-24 induction, even more these responses appeared to be dependent of specific protein kinase C isoforms and independent of inhibition of adenylyl cyclase. A similar coupling of CB2 to a mitogenic pathway and to the regulation of Krox-24 expression was also observed in human promyelocytic cells HL60. Taken together, these findings provide evidence for a functional role of the CB2 receptor in gene induction mediated by the MAP kinase network.

A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions.

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.