ABSTRACT
Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR T-cells were specifically activated by CD19-positive tumor cell lines and primary chronic lymphocytic leukemia cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine-based CARs.
Subject(s)
Antigens, CD19/immunology , Burkitt Lymphoma/therapy , Immunotherapy, Adoptive/methods , Recombinant Fusion Proteins/immunology , Single-Chain Antibodies/immunology , T-Lymphocytes/transplantation , Animals , Burkitt Lymphoma/pathology , Cell Line, Tumor , Female , Gene Library , HEK293 Cells , Humans , K562 Cells , Mice , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Recombinant Fusion Proteins/genetics , Single-Chain Antibodies/genetics , Species Specificity , Transduction, Genetic , Xenograft Model Antitumor AssaysABSTRACT
The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1' group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.
Subject(s)
Acetylene/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Metalloendopeptidases/antagonists & inhibitors , ortho-Aminobenzoates/chemistry , ADAM Proteins , ADAM17 Protein , Crystallography, X-Ray , Hydroxamic Acids/chemistry , Molecular Structure , Structure-Activity Relationship , Sulfonamides/chemistry , ortho-Aminobenzoates/pharmacologyABSTRACT
Potent and selective bicyclic heteroaryl hydroxamic acid MMP and TACE inhibitors were synthesized by a novel convergent route. Selectivity and efficacy versus MMPs and TACE could be controlled by appropriate substitution on the scaffolds and by variation of the P1' group. Select compounds were found to be effective in in vivo models of arthritis.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Animals , Arthritis/drug therapy , Arthritis/pathology , Biological Availability , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cartilage/drug effects , Cartilage/pathology , Cattle , Disease Models, Animal , Enzyme Inhibitors/chemical synthesis , Hydroxamic Acids/chemical synthesis , Mice , Rats , Structure-Activity RelationshipABSTRACT
Etanercept, a recombinant human tumor necrosis factor (TNF) inhibitor that binds both soluble and cell-bound TNF, has been shown to reduce disease activity and inhibit joint destruction when administered to patients with rheumatoid arthritis (RA). Because TNF receptors are found on many types of cells that modulate the immune response, we evaluated the general immune function of a subset of RA patients in a blinded clinical study. No significant differences were seen between patients treated with etanercept or placebo in the surface antigen phenotypes of peripheral blood leukocytes, T cell proliferative responses, neutrophil function, delayed-type hypersensitivity (DTH) reactions, serum immunoglobulin levels, or incidence of infections. Although this observational study was relatively small and could detect only major changes in immunological status, the stability of immune function over time in patients receiving etanercept corroborates the findings in clinical studies, which suggest that etanercept does not alter overall global immune function.
Subject(s)
Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Immunoglobulin G/therapeutic use , Receptors, Tumor Necrosis Factor/therapeutic use , Adult , Aged , Arthritis, Rheumatoid/immunology , Double-Blind Method , Etanercept , Humans , Immunoglobulins/blood , Immunophenotyping , Infections/epidemiology , Lymphocyte Activation , Middle Aged , Neutrophils/physiology , T-Lymphocytes/immunologyABSTRACT
The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.
Subject(s)
Acetylene/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , ortho-Aminobenzoates/chemistry , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Structure-Activity RelationshipABSTRACT
Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.
Subject(s)
Amines/chemistry , Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Animals , Binding Sites , Collagenases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hydroxamic Acids/chemistry , Interleukin-1 , Interleukin-1beta , Magnetic Resonance Spectroscopy , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 9/metabolism , Metalloendopeptidases , Mice , Models, Molecular , Osteoarthritis/drug therapy , Rats , Structure-Activity Relationship , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistryABSTRACT
A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, MMP-13, and TACE was prepared and evaluated. Selective inhibitors of MMP-9, MMP-13, and TACE were identified, including the potent, orally active MMP-13 inhibitor 4p.
Subject(s)
Enzyme Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors , ortho-Aminobenzoates/pharmacology , ADAM Proteins , ADAM17 Protein , Collagenases/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Matrix Metalloproteinase 13 , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinases/metabolism , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Structure-Activity Relationship , ortho-Aminobenzoates/chemical synthesis , ortho-Aminobenzoates/chemistrySubject(s)
Heart Failure/drug therapy , Immunoglobulin G/therapeutic use , Immunologic Factors/therapeutic use , Receptors, Tumor Necrosis Factor/therapeutic use , Antigens, CD/therapeutic use , Etanercept , Humans , Immunoglobulin G/chemistry , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor, Type II , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
To determine the roles of the type 1 tumor necrosis factor (TNF) receptor (TNFR1) in lung inflammation and antibacterial defense, we exposed transgenic mice lacking TNFR1 [TNFR1(-/-)] and wild-type control mice to aerosolized lipopolysaccharide or Pseudomonas aeruginosa. After LPS, bronchoalveolar lavage fluid (BALF) from TNFR1(-/-) mice contained fewer neutrophils and less macrophage inflammatory protein-2 than BALF from control mice. TNF-alpha, interleukin-1beta, and total protein levels in BALF as well as tissue intercellular adhesion molecule-1 expression did not differ between the two groups. In contrast, lung inflammation and bacterial clearance after infection were augmented in TNFR1(-/-) mice. BALF from infected TNFR1(-/-) mice contained more neutrophils and TNF-alpha and less interleukin-1beta and macrophage inflammatory protein-2 than that from control mice, but protein levels were similarly elevated in both groups. Lung inflammation and bacterial clearance were also augmented in mice lacking both TNF receptors. Thus TNFR1 facilitates neutrophil recruitment after inhalation of lipopolysaccharide, in part by augmenting chemokine induction. In contrast, TNFR1 attenuates lung inflammation in response to live bacteria but does not contribute to increased lung permeability and is not required for the elimination of P. aeruginosa.
Subject(s)
Endotoxins/administration & dosage , Pneumonia, Bacterial/microbiology , Pseudomonas Infections , Receptors, Tumor Necrosis Factor/physiology , Administration, Inhalation , Animals , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Chemokine CXCL2 , Chemotaxis, Leukocyte , Cytokines/analysis , Intercellular Adhesion Molecule-1/analysis , Interleukin-1/analysis , Leukocyte Count , Mice , Mice, Inbred C57BL , Mice, Transgenic , Monokines/analysis , Neutrophils , Pneumonia, Bacterial/metabolism , Pneumonia, Bacterial/pathology , Proteins/analysis , Receptors, Tumor Necrosis Factor/deficiency , Receptors, Tumor Necrosis Factor/genetics , Tumor Necrosis Factor-alpha/analysisABSTRACT
Mouse NK cells may use both cytokine, e.g. IFN-gamma, tumor necrosis factor (TNF)-alpha and IL-12, and cytotoxic, e.g. perforin and Fas-FasL, pathways to reject incompatible bone marrow cell (BMC) grafts. To begin a dissection of these two major pathways, mice bearing deletional mutations of IFN-gamma, TNF-RI/II or perforin, or mice treated with mAb to IL-12, IFN-gamma or NK1.1 were irradiated and challenged with class I-deficient BMC grafts, a system in which only NK cells are the effector cells. Proliferation of the donor-derived cells was judged in terms of splenic incorporation of [125I]iododeoxyuridine 5 or 7 days after cell transfer. All of these mice maintained in a specific pathogen-free (s.p.f.) environment were able to reject the BMC, except those treated with anti-NK1.1 mAb. However, perforin deficient mice maintained in a conventional breeding facility failed to reject class I (Tap-1)-deficient marrow cells. Transfer of mice from the pathogen-free to the conventional facility resulted in a slow and incomplete loss of the ability to reject marrow cells. Thus, the breeding colony environment can elicit otherwise undetectable defects in the rejection ability of perforin-deficient NK cells. This report will hopefully alert those investigators who have only studied immune gene knockout mice in s.p.f. facilities and found no significant abnormalities.
Subject(s)
Bone Marrow Transplantation/immunology , Cytokines/metabolism , Cytotoxins/metabolism , Environmental Exposure , Graft Rejection/immunology , Killer Cells, Natural/immunology , Animals , Antibodies, Monoclonal , Cytokines/immunology , Cytotoxins/immunology , Housing, Animal , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Specific Pathogen-Free OrganismsABSTRACT
The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.
Subject(s)
Antigens, CD/metabolism , Antigens, CD/physiology , Inflammation/immunology , Receptors, Tumor Necrosis Factor/metabolism , Receptors, Tumor Necrosis Factor/physiology , Acute-Phase Reaction/genetics , Acute-Phase Reaction/immunology , Animals , Antigens, CD/blood , Antigens, CD/genetics , Cell Differentiation/genetics , Cell Differentiation/immunology , Crosses, Genetic , Disease Models, Animal , Endotoxemia/genetics , Endotoxemia/immunology , Endotoxemia/mortality , Farmer's Lung/genetics , Farmer's Lung/immunology , Farmer's Lung/pathology , Female , Immunity, Innate , Inflammation/genetics , Listeriosis/immunology , Lymphocyte Subsets/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred Strains , Mice, Knockout , Receptors, Tumor Necrosis Factor/blood , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Thymus Gland/cytology , Thymus Gland/growth & development , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Pro-TNF alpha, Steel factor, type II IL-1R and IL-2R alpha were expressed in COS-7 cells and the generation of their soluble forms was examined. The release of all four proteins was strongly stimulated by the phorbol ester PMA and completely blocked by a hydroxamate-based inhibitor of metalloproteases. COS-7 cell membranes were found to cleave various synthetic pro-TNF alpha peptides with the same specificity as a partially purified TNF alpha converting enzyme purified from human monocytic cells, suggesting that the same enzyme may be responsible for at least some of the COS-7 cell shedding activity.
Subject(s)
Membrane Proteins/metabolism , Animals , COS Cells , Cell Line , Dipeptides/pharmacology , Humans , Hydroxamic Acids/pharmacology , Receptors, Interleukin/metabolism , Stem Cell Factor/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Tumor necrosis factor-alpha is released from cells by a proteolytic cleavage. Previous work suggested that a specific, non-matrix metalloproteinase carries out this cleavage, but matrix metalloproteinases have also been implicated. In this paper, we report that none of the matrix metalloproteinases tested cleaved peptide substrates as specifically as the non-matrix metalloproteinase. A matrix metalloproteinase did process tumor necrosis factor-alpha extracted from COS cells, but neither tissue inhibitor of metalloproteinases-1 nor -2 blocked tumor necrosis factor-alpha processing by human monocytes. Moreover, tissue inhibitor of metalloproteinases-1 had at most a partial effect on the in vivo release of the cytokine in mice. We conclude that a non-matrix metalloproteinase is the major physiological tumor necrosis factor-alpha convertase.
Subject(s)
Glycoproteins/pharmacology , Metalloendopeptidases/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , ADAM Proteins , ADAM17 Protein , Animals , CHO Cells , Cell Line , Cells, Cultured , Cricetinae , Humans , Mice , Mice, Inbred BALB C , Protein Processing, Post-Translational , Recombinant Proteins/pharmacology , Substrate Specificity , Tissue Inhibitor of Metalloproteinases , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL-12, but not IL-2, in these cultures by approximately 90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-gamma and granulocyte/macrophage colony-stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L.
Subject(s)
CD40 Antigens/pharmacology , Interleukin-12/biosynthesis , Macrophages, Peritoneal/metabolism , Membrane Glycoproteins/pharmacology , T-Lymphocytes, Helper-Inducer/immunology , Animals , Antibodies, Monoclonal/pharmacology , CD40 Ligand , Cell-Free System/immunology , Clone Cells , Cytokines/pharmacology , Drug Interactions/immunology , Female , Lymphocyte Activation , Male , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Recombinant Proteins/pharmacology , T-Lymphocytes/immunologyABSTRACT
Tumor necrosis factor alpha (TNFa) has been shown to be the primary cytokine responsible for the OKT3-induced acute clinical syndrome (OKT3-ACS). Recombinant human soluble tumor necrosis factor receptor (TNFR:Fc) is a dimer of the p80 TNF receptor, which binds both TNFa and lymphotoxin (LT). Renal allograft recipients undergoing OKT3 therapy for steroid-resistant rejection were randomized to receive OKT3 alone or in combination with TNFR:Fc to determine its safety and efficacy in decreasing the severity of OKT3-ACS and in restoring renal function. Six of 12 patients were given TNFR:Fc prior to each of the first two injections of OKT3. All patients were monitored for manifestations of OKT3-ACS and changes in renal function. In addition, serial serum samples were assayed for TNFa and TNFR:Fc levels (ELISA) and TNFa bioactivity (L929). No adverse side effects were identified in patients receiving TNFR:Fc. Patients treated with TNFR:Fc had significantly fewer symptoms by day 2 of OKT3, and had a lower overall incidence of chills and arthralgias. Renal dysfunction reversed within 24 hr in the TNFR:Fc-treated group in contrast to the 48-72-hr delay in the control group. Antigenic TNFa levels increased in the control group from < 10 pg/ml pre OKT3 to a mean peak level of 30 +/- 13 pg/ml on day 1 and decreased to pretreatment levels by day 2. TNFR:Fc-treated patients had a mean peak TNFa level of 235 +/- 135 pg/ml, suggesting a carrier effect of TNFR:Fc. In contrast, bioactivity was barely detectable (mean 20 +/- 14 pg/ml) in the day 1 samples from TNFR:Fc-treated patients, whereas significant bioactivity (peak mean 60 +/- 35 pg/ml) was detected in sera from control patients. TNF receptor levels reached 600 ng/ml in treated patients and remained elevated for up to 18 days confirming the long half-life of TNFR:Fc. This phase 1 trial demonstrates that TNFR:Fc is well tolerated and may limit the severity of OKT3-ACS. The most significant observation was a more rapid improvement in renal function in the TNFR:Fc-treated patients. The absence of TNFa bioactivity indicates that TNFR:Fc functions as a TNF antagonist. Further evaluation of higher doses of TNFR:Fc in OKT3-treated patients is currently in progress.
Subject(s)
Graft Rejection/prevention & control , Immunosuppressive Agents/adverse effects , Kidney Transplantation , Muromonab-CD3/adverse effects , Receptors, Tumor Necrosis Factor , Acute Disease , Humans , Recombinant Proteins/therapeutic use , Syndrome , Transplantation, HomologousABSTRACT
Many cytokines and soluble cytokine receptors are generated by limited proteolysis of membrane-bound precursors. We have examined the ability of the recently described metalloprotease inhibitor, TNF-alpha protease inhibitor (TAPI), and other protease inhibitors to modulate shedding. The membrane-bound forms of the ligands TNF-alpha and CSF-1, the p60 TNFR and the IL-6R, were expressed in COS-7 cells. As expected, TAPI blocked the spontaneous and PMA-induced release of TNF-alpha from transfected cells. Interestingly, TAPI also inhibited the release of soluble forms of p60 TNFR and IL-6R in COS-7 cells. However, the processing of CSF-1, which also requires proteolytic cleavage of a membrane protein, was not affected. The ability of TAPI to inhibit shedding was unique, since several other classes of protease inhibitors, including three other metalloprotease inhibitors, did not inhibit shedding of IL-6R. To determine whether TAPI would prevent shedding under more physiologic conditions, we demonstrated that TAPI was able to prevent unstimulated and PMA-induced release of the soluble forms of TNF-alpha, p60 TNFR, and IL-6R from the monocytic cell line, THP-1, and from human peripheral blood monocytes. In addition, TAPI was able to inhibit LPS-induced shedding of the p60 TNFR and TNF-alpha from monocytes. In summary, our results indicate that a metalloprotease or group of related metalloproteases is responsible for the proteolytic cleavage of several cell surface proteins.
Subject(s)
Antigens, CD/metabolism , Dipeptides/pharmacology , Hydroxamic Acids/pharmacology , Metalloendopeptidases/metabolism , Protease Inhibitors/pharmacology , Receptors, Interleukin/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Cell Line , Humans , Lipopolysaccharides/pharmacology , Macrophage Colony-Stimulating Factor/drug effects , Macrophage Colony-Stimulating Factor/metabolism , Metalloendopeptidases/antagonists & inhibitors , Monocytes , Receptors, Interleukin-6 , Tetradecanoylphorbol Acetate/pharmacology , Transfection , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/metabolismABSTRACT
TNF is synthesized as a 26-kD membrane-anchored precursor and is proteolytically processed at the cell surface to yield the mature secreted 17-kD polypeptide. The 80-kD tumor necrosis factor (TNF) receptor (TNFR80) is also proteolytically cleaved at the cell surface (shed), releasing a soluble ligand-binding receptor fragment. Since processing of TNF and TNFR80 occurs concurrently in activated T cells, we asked whether a common protease may be involved. Here, we present evidence that a recently described inhibitor of TNF processing N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl)L- 3-(2'naphthyl)- alanyl-L-alanine, 2-aminoethyl amide (TAPI) also blocks shedding of TNFR80, suggesting that these processes may be coordinately regulated during T cell activation. In addition, studies of murine fibroblasts transfected with human TNFR80, or a cytoplasmic deletion form of TNFR80, reveal that inhibition of TNFR80 shedding by TAPI is independent of receptor phosphorylation and does not require the receptor cytoplasmic domain.
Subject(s)
Dipeptides/pharmacology , Hydroxamic Acids/pharmacology , Metalloendopeptidases/antagonists & inhibitors , Receptors, Tumor Necrosis Factor/antagonists & inhibitors , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Humans , Lymphocyte Activation , Metalloendopeptidases/physiology , Mice , Receptors, Tumor Necrosis Factor/metabolism , Transfection , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Tumour necrosis factor (tumour necrosis factor-alpha/cachectin) plays a critical role in certain physiological defensive responses but causes severe damage to the host organism when produced in excess. There are two forms of tumour necrosis factor, a type II membrane protein of relative molecular mass 26,000 (26K) and a soluble, 17K form generated from the cell-bound protein by proteolytic cleavage. The two forms of tumour necrosis factor and lymphotoxin-alpha (tumour necrosis factor-beta/lymphotoxin), a related protein, have similar but apparently not identical biological activities. A therapeutic agent which inhibited the release of tumour necrosis factor, but did not reduce the cell-associated activity or the level of lymphotoxin-alpha, might preserve the benefits of these cytokines while preventing tumour necrosis factor-induced damage. Here we describe a potent inhibitor of tumour necrosis factor processing and report that it protects mice from a lethal dose of endotoxin.
Subject(s)
Protein Processing, Post-Translational/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Amino Acid Sequence , Animals , Cell Line , Humans , Hydroxamic Acids/pharmacology , Lymphotoxin-alpha/metabolism , Metalloendopeptidases/antagonists & inhibitors , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Recombinant Proteins , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/toxicityABSTRACT
T cell-dependent regulation of B cell growth and differentiation involves an interaction between CD40, a B cell surface molecule, and the CD40 ligand (CD40L) which is expressed on activated CD4+ T cells. In the current study, we show that recombinant membrane-bound murine CD40L induces B cells to express costimulatory function for the proliferation of CD4+ T cells. CD40L- or lipopolysaccharide (LPS)-activated, but not control-cultured B cells were strong costimulators of anti-CD3 or alloantigen-dependent T cell responses. The molecular interactions responsible for the increased costimulatory functions were examined by analyzing the activated B cells for changes in the expression of two costimulatory molecules, B7 and heat-stable antigen (HSA), as well as by the use of antagonists of B7 and HSA (CTLA4.Fc and 20C9, respectively). The expression of both B7 and HSA was enhanced on B cells activated with LPS. As observed in previous studies, the costimulatory activity of the LPS-activated B cells was dependent on both B7 and HSA and was completely inhibited in the presence of a combination of CTLA4.Fc and 20C9. In contrast, activation of B cells with CD40L induced the expression of B7 but did not enhance the expression of HSA. In addition the costimulatory activity of the CD40L-activated B cells was partially, but not completely, inhibited by the combination of CTLA4.Fc and 20C9. These results demonstrate that CD40L regulates costimulatory function of B cells in part by inducing the expression of B7 and suggest that CD40L-activated B cells express an additional costimulatory activity that is not associated with LPS-activated B cells.
Subject(s)
B-Lymphocytes/physiology , Membrane Glycoproteins/physiology , Animals , Antigens, Differentiation/biosynthesis , B7-1 Antigen/biosynthesis , Base Sequence , CD40 Ligand , Cell Adhesion Molecules/biosynthesis , Female , Histocompatibility Antigens Class II/biosynthesis , Intercellular Adhesion Molecule-1 , Lipopolysaccharides/immunology , Lymphocyte Activation/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Recombinant Proteins , T-Lymphocytes/physiologyABSTRACT
Two forms (monomeric or dimeric) of the extracellular, ligand-binding portion of the human p80 cell-surface receptor for TNF were used to antagonize TNF activity in vitro and in vivo. The dimeric sTNFR:Fc molecule was a more potent inhibitor of TNF than the monomeric sTNFR (50 to 1000x), as assessed in vitro by inhibition of TNF binding or bioactivity and in vivo by protection of mice from an otherwise lethal injection of LPS. Surprisingly, the dimeric sTNFR:Fc construct demonstrated a beneficial effect even when administered 3 h after a lethal LPS injection (i.e., after serum TNF levels had peaked and receded). To study the mechanism by which the soluble TNFR functions in vivo, serum TNF levels were examined in mice given LPS in the presence or absence of soluble receptor. Administration of a mortality-reducing dose of sTNFR:Fc ablated the rise in serum TNF bioactivity that normally occurs in response to LPS. However, TNF bioactivity was revealed in these "TNF-negative" serum samples when the L929 bioassay was modified by inclusion of a mAb that blocks the binding of murine TNF to the human soluble TNFReceptor. These results indicate that the absence of direct cytolytic activity in the L929 assay was caused by neutralization of TNF, rather than to an absence of TNF in the serum. Moreover, administration of either monomeric sTNFR or low doses of dimeric sTNFR:Fc actually resulted in increased serum TNF levels compared to mice given LPS but no soluble receptor. However, these "agonistic" doses of soluble receptor did not lead to increased mortality when an LD60 dose of LPS was given. Thus, dimeric sTNFR are effective inhibitors of TNF and under some circumstances function simultaneously as both TNF "carriers" and antagonists of TNF biologic activity.