Sigma receptor ligands: applications in inflammation and oncology.

Sigma (sigma) receptors were initially proposed as a subtype of opiate receptors, and bind several psychoactive compounds. They are classified into sigma 1 (sigma1) and sigma 2 (sigma2) subtypes. The characterization of these subunits, and the discovery of new specific sigma receptor ligands, demonstrated that sigma receptors belong to a specific entity distinct from opiate receptors.

Genomic and functional changes induced by the activation of the peripheral cannabinoid receptor CB2 in the promyelocytic cells HL-60. Possible involvement of the CB2 receptor in cell differentiation.

The function of the peripheral cannabinoid receptor (CB2), which is mainly expressed on hematopoietic cells, remains an enigma. In an attempt to decipher its role, we used Affymetrix DNA chips to investigate the gene expression profile of the promyelocytic cells HL-60 transfected with the CB2 receptor and activated with the cannabinoid agonist CP 55,940. Agonist exposure of these cells led to an activation of a mitogen-activated protein kinase cascade and a receptor desensitization, indicating a functional coupling of the transfected receptors.

Stimulation of peripheral cannabinoid receptor CB2 induces MCP-1 and IL-8 gene expression in human promyelocytic cell line HL60.

Using the recently developed methodology of nucleic acid microarrays spotted with specific cDNAs probes belonging to different gene families, we showed for the first time that nanomolar concentrations of the cannabinoid ligand CP-55940 upregulated the expression of two different members of the chemokine gene family: the alpha-chemokine interleukin-8 (IL-8) and the beta-chemokine monocyte chemotactic protein-1 (MCP-1), in the promyelocytic cell line HL60 transfected with peripheral cannabinoid receptors (CB2). These genomic modulations observed on large-scale cDNA arrays were first confirmed by Northern blot studies. Furthermore, ELISA evaluations in culture supernatants indicated that the cannabinoid-induced activation of these two chemokine genes was followed by enhanced expression and secretion of the corresponding proteins.

Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.

Two subtypes of G-protein-coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins.

The endogenous cannabinoid anandamide is a lipid messenger activating cell growth via a cannabinoid receptor-independent pathway in hematopoietic cell lines.

The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype.

SR 144528, the first potent and selective antagonist of the CB2 cannabinoid receptor.

Based on both binding and functional data, this study introduces SR 144528 as the first, highly potent, selective and orally active antagonist for the CB2 receptor. This compound which displays subnanomolar affinity (Ki = 0.6 nM) for both the rat spleen and cloned human CB2 receptors has a 700-fold lower affinity (Ki = 400 nM) for both the rat brain and cloned human CB1 receptors.

Effect of substance P on cytokine production by human astrocytic cells and blood mononuclear cells: characterization of novel tachykinin receptor antagonists.

Substance P (SP) has been reported to induce inflammatory cytokine production in human neuroglial cells and peripheral lymphoid cells as well. In order to evaluate the potency of novel non-peptide antagonists of the tachykinin receptors as inhibitors of SP-induced cytokines, we used the astrocytoma cell line U373MG and blood mononuclear cells as models of central and peripheral SP-target cells, respectively. In the first part of this study, we showed that SR 140333, an NK1 tachykinin receptor antagonist, was able to inhibit strongly the SP-induced production of interleukin (IL)-6 and IL-8 in the astrocytoma cell line.

A sigma ligand, SR 31747A, potently modulates Staphylococcal enterotoxin B-induced cytokine production in mice.

Sigma receptors originally described in distinct regions of the central nervous system are expressed on cells of the immune system. A sigma ligand, SR 31747A, was observed here to inhibit in vitro the Staphylococcal enterotoxin B (SEB)-driven lymphocyte proliferation. In mice, the drug confers a potent protection against the lethality induced by SEB, stimulates the SEB-induced serum release of interleukin (IL)-10 and inhibits at the same time the systemic release of IL-2, IL-4, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6 and tumour necrosis factor-alpha (TNF-alpha).

Echinomycin suppresses the pyrogenic effects of endotoxin and interleukin-1 beta in human endothelial cells and peripheral blood mononuclear cells.

Endotoxin (LPS) and interleukin-1 beta (IL-1 beta) increased in a dose-dependent manner the expression of tissue factor, an ubiquitous membrane-anchored glycoprotein that initiates blood coagulation at the surface of human umbilical vein endothelial cells (HUVEC and human peripheral blood mononuclear cells (PBMC). Echinomycin, a cyclic octapeptide of microbial origin strongly inhibited LPS- and IL-1 beta-induced tissue factor expression in HUVEC and PBMC with IC50 values in the subnanomolar range at the same time it reduced LPS and IL-1 beta-induced expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on HUVEC (IC50 = 0.4 +/- 0.

Enhancement of endotoxin-induced interleukin-10 production by SR 31747A, a sigma ligand.

SR 31747A is a new sigma ligand eliciting immunosuppressive and anti-inflammatory properties. Here, we show that SR 31747A greatly enhances lipopolysaccharide (LPS)-induced systemic release of interleukin (IL)-10, while it inhibits the secretion of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. In line with this finding, we also show by using quantitative reverse transcription-polymerase chain reaction analysis that SR 31747A increased LPS-induced IL-10 mRNA accumulation in spleen cells, whereas the level of both TNF-alpha and IFN-gamma mRNA was dramatically decreased.

Cannabinoids enhance human B-cell growth at low nanomolar concentrations.

This study examined the effect of cannabinoid ligands on human tonsillar B-cells activated either through cross-linking of surface immunoglobulins or ligation of the CD40 antigen. The two synthetic cannabinoids, CP55,940 and WIN55212-2, as well as delta 9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, caused a dose-dependent increase of B-cell proliferation and displayed EC50 at low nanomolar concentrations. This cannabinoid-induced enhancing activity was inhibited by pertussis toxin which suggested a G-protein-coupled receptor process.

In vivo inhibition of endotoxin-induced pro-inflammatory cytokines production by the sigma ligand SR 31747.

In previous studies, the authors demonstrated that the new sigma ligand, cis-N-cyclohexyl-N-ethyl-3-(3-chloro-4-cyclohexyl-phenyl) propen-2-ylamine hydrochloride (SR 31747), elicited a suppressive effect on immune responses through the sigma receptor expressed on lymphocytes. Here the effect of SR 31747 on the proinflammatory cytokine production by endotoxin-activated macrophages is examined. In vivo, SR 31747 dramatically blocked lipopolysaccharide-induced production of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha in a dose-dependent manner (ED50, 2 mg/kg).

Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4.

Interleukin-13 (IL-13) is a recently described human lymphokine which is produced by activated T-cells. Its effect on the production of IL-6 by normal keratinocytes and keratinocyte cell lines of human origin was studied and compared to that of IL-4. IL-13, similarly to IL-4, stimulated IL-6 expression by these cells in a dose- and time-dependent manner.

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.

Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses.

The discovery of new cytokines normally relies on a prior knowledge of at least one of their biological effects, which is used as a criterion either for the purification of the protein or for the isolation of the complementary DNA by expression cloning. However, the redundancy of cytokine activities complicates the discovery of novel cytokines in this way, and the pleiotropic nature of many cytokines means that the principal activities of a new cytokine may bear little relation to that used for its isolation. We have adopted an alternative approach which relies on differential screening of an organized subtracted cDNA library from activated peripheral blood mononuclear cells, using the inducibility of lymphokine messenger RNAs by anti-CD28 as a primary screening criterion.

Golgi vacuolization and immunotoxin enhancement by monensin and perhexiline depend on a serum protein. Implications for intracellular trafficking.

Vacuole formation around the Golgi and immunotoxin enhancement induced by low doses of the ionophore monensin were inhibited by 50% human plasma (final concentration), whereas the lysosomal pH increase remained unaffected. Immunotoxin enhancement by the Ca2+ antagonist perhexiline was also inhibited by plasma. The inhibiting factor was present in different species and highly concentration-dependent.

Biologically active A-chain of the plant toxin ricin expressed from a synthetic gene in Escherichia coli.

To assess the biological activity and pharmacokinetic properties of nonglycosylated ricin A-chain (RA), we have obtained the polypeptide following expression of a synthetic 842-bp RA gene in Escherichia coli. Expression of the gene was carried out using the phage T5 PN25 promoter fused to the E. coli lac operator.

Specific elimination of alloreactive T cells by an anti-interleukin-2 receptor B chain-specific immunotoxin.

Graft-versus-host disease and graft rejection remain the two principal causes of morbidity and mortality after major-histocompatibility-complex-mismatched bone marrow transplantation. Human and animal models suggest that both CD4+ and CD8+ T cell subsets present in the donor inoculum are responsible for their initiation. Since the human mixed lymphocyte culture (MLC) and the HLA-restricted cytotoxicity may reflect cellular interactions occurring during GVHD and graft rejection, inhibitions of these responses may represent useful approaches for screening functional T cell depletion in experimental bone marrow transplantation studies.

Donor bone marrow treatment with T101 Fab fragment-ricin A-chain immunotoxin prevents graft-versus-host disease.

Thirty-eight patients with haematological malignancies were treated with bone marrow transplantation using histocompatible immunotoxin T cell-depleted marrow siblings. All patients received conventional postgraft immunosuppression (methotrexate and/or cyclosporin A). Donor bone marrow was treated ex vivo with T101 Fab fragment coupled to ricin A-chain (T101 Fab-RTA) at a concentration of 10(-8) M of A-chain in association with NH4Cl (2 x 10(-2) M) in pH adjusted (7.

Antigenic modulation induced by four monoclonal antibodies adsorbed on gold particles (specificity anti-CD4, anti-CD5, anti-CD7, and anti-150-kDa antigen): relationship between modulation and cytotoxic activity of immunotoxins.

The present study concerns the antibody-induced antigenic modulation of CD4, CD5, CD7, and 150-kDa antigens present on cells of the CCRF-CEM human T line. The immunogold electron microscopy method was used, and it was found that the entry routes associated with the various antigen-antibody complexes were different. Thus, the anti-CD7 monoclonal antibody (MoAb) was frequently internalized via the coated structures of the cell membrane, whereas anti-CD5 MoAb was rarely internalized via those structures and anti-CD4 and anti-150-kDa antigens never used this route.

Sensitivity of fresh leukemic cells to T101 ricin A-chain immunotoxin: a comparative study between Fab fragment and whole Ig conjugates.

We compared the cell killing potency of a whole Ig ricin A-chain immunotoxin (T101 IgG-RTA) against its Fab fragment counterpart (T101 Fab-RTA) on both CEM cells and fresh malignant lymphoid cells. A dye exclusion assay (DEA), was used to evaluate the kinetics of leukaemia cell viability mediated in vitro by each immunotoxin (IT). This study found that in the absence of ammonium chloride (NH4Cl), used as an enhancer agent, T101 Fab-RTA was significantly more toxic to both CEM and fresh leukaemia cells than T101 IgG-RTA.

Effects of an anti HLA-DR immunotoxin on leukaemia cells and hematopoietic progenitors.

An anti HLA-class II immunotoxin has been prepared by coupling to ricin A-chain (RTA) and anti-DR-DP monoclonal antibody (2G5-MoAb). Protein synthesis inhibition assays showed that 2G5-RTA immunotoxin is: highly cytotoxic to B-cell lymphoid neoplastic cells, and variable so to ALL cells, while AML cell lines display a generally poor susceptibility. Toxicity of 2G5-RTA on normal hematopoietic cells (HPC) was found to be dose-dependent, and to increase significantly with the addition of NH4Cl, used as an activating agent.

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins.

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used.

T-lymphocyte killing by T101-ricin A-chain immunotoxin: pH-dependent potentiation with lysosomotropic amines.

To maximize T-lymphocyte killing with anti-pan-T-lymphocyte immunotoxin (IT), prepared by linking ricin A-chain to monoclonal antibody (MoAb) T101 (T101-RTA IT), we have established the nature and the extent of parameters that influence the sensitivity of T lymphocytes to the IT. We showed that peripheral blood T lymphocytes, which are much less susceptible than malignant T cells to the T101-RTA IT, could become highly sensitive to the IT when used in conjunction with NH4Cl. However, enhancement of the IT by NH4Cl only occurred when the pH rose above neutrality.

Rationale for the selection of ricin A-chain anti-T immunotoxins for mature T cell depletion.

A series of 25 different ricin A-chain immunotoxins (IT) were prepared with monoclonal antibodies reacting with several T cell antigens belonging to different clusters of differentiation (CD) to select the most appropriate immunotoxins (IT) for mature T cell depletion. Our screening procedure was performed in 2 steps. First, IT were evaluated using protein synthesis inhibition assay on clonogenic malignant cells in order to determine the most active IT for a given CD.