ABSTRACT
The CXC chemokine CXCL13, known as BCA-1 (B cell-attracting chemokine 1) or BLC (B-lymphocyte chemoattractant), has been identified as an efficacious attractant selective for B lymphocytes. The chemokine receptor BLR1 (Burkitt's lymphoma receptor 1)/CXCR5 expressed by all mature B cells has to date been identified as the only known receptor for BCA-1. As the loss of the BLR1/CXCR5 receptor is sufficient to disrupt organization of follicles in spleen and Peyer's patches, BCA-1 may act as a B cell homing chemokine. Nonetheless, BCA-1 has not been tested against all known chemokine receptors. In this study, we report that human BCA-1 competes with radiolabeled interferon gamma (IFN-gamma) inducible protein 10 (IP-10) for binding to the human CXCR3 receptor expressed in Ba/F3 and 293EBNA cell lines. Furthermore, human BCA-1 is an efficacious attractant for human CXCR3 transfected cells; BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3. In these cells, as in human B lymphocytes expressing CXCR5, BCA-1 does not induce a calcium flux. Indeed, BCA-1 attenuates the calcium flux induced by IP-10. In addition, human BCA-1 is an agonist in stimulating GTP gamma S binding. Together these data suggest that human BCA-1 is a specific and functional G-protein-linked chemotactic ligand for the human CXCR3 receptor. The biological significance of this new finding is supported by our recent observation that human BCA-1 induces chemotaxis of activated T cells and the BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3.
Subject(s)
B-Lymphocytes/metabolism , Chemokines, CXC/metabolism , Chemokines, CXC/physiology , Receptors, Chemokine/agonists , Animals , Antibodies, Monoclonal/metabolism , Calcium/metabolism , Cell Line , Cell Membrane/metabolism , Cell Separation , Chemokine CXCL10 , Chemokine CXCL13 , Chemokines/metabolism , Chemotaxis , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Interferon-gamma/metabolism , Ligands , Mice , Protein Binding , Rats , Receptors, CXCR3 , Receptors, CXCR5 , Receptors, Cytokine/metabolism , Time Factors , TransfectionABSTRACT
Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (-/-) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8(-/)- mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo.
Subject(s)
Eosinophils/immunology , Hypersensitivity/immunology , Receptors, Chemokine/deficiency , Th2 Cells/immunology , Administration, Inhalation , Animals , Antigens/administration & dosage , Antigens/immunology , Cockroaches/immunology , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Immunologic , Eosinophils/cytology , Granuloma/immunology , Granuloma/pathology , Hypersensitivity/genetics , Hypersensitivity/pathology , Immunity, Cellular/genetics , Immunity, Cellular/immunology , Injections, Subcutaneous , Interleukin-5/blood , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin/administration & dosage , Ovalbumin/immunology , Ovum/immunology , RNA, Messenger/metabolism , Receptors, CCR8 , Receptors, Chemokine/genetics , Schistosoma mansoni/immunology , Th1 Cells/immunologyABSTRACT
The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-gamma), and I-TAC (interferon-inducible T cell alpha chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [(35)S]GTP gamma S binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. (125)I-IP-10 and (125)I-I-TAC bound hCXCR3 with high affinity, although the (125)I-I-TAC B(max) value in saturation bindings was 7- to 13-fold higher than that measured with (125)I-IP-10. Coincubation with unlabeled chemokines decreased (125)I-IP-10 binding with a single discernible affinity. However, with (125)I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC(50) values did not increase predictably with increased (125)I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated (125)I-IP-10 binding but only decreased (125)I-I-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TAC- and IP-10-stimulated [(35)S]GTP gamma S binding. The antibody attenuated [(35)S]GTP gamma S binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a >75-fold increase in (125)I -IP-10 concentration did not increase the I-TAC binding IC(50) value, and I-TAC increased the dissociation rate of (125)I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.
Subject(s)
Chemokines, CXC/metabolism , Receptors, Chemokine/metabolism , Animals , Antibodies/pharmacology , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Binding, Competitive/drug effects , Binding, Competitive/physiology , Cell Membrane/metabolism , Cells, Cultured , Chemokine CXCL10 , Chemokine CXCL11 , Chemokines/pharmacology , Dose-Response Relationship, Drug , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Humans , Ligands , Lymphocytes/cytology , Lymphocytes/metabolism , Mice , Receptors, CXCR3 , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/drug effectsABSTRACT
Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyer's patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Movement/immunology , Dendritic Cells/immunology , Immunity, Mucosal , Receptors, Chemokine/immunology , Animals , CD11 Antigens/immunology , Dendritic Cells/pathology , Mice , Mice, Knockout , Receptors, CCR6ABSTRACT
Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.
Subject(s)
Interferon-gamma/metabolism , Receptors, Interferon/metabolism , Signal Transduction , Base Sequence , Biopolymers , Cell Line , Chromatography, Gel , DNA Primers , Dimerization , Humans , Interferon-gamma/chemistry , Protein Binding , Interferon gamma ReceptorABSTRACT
Human herpesvirus 8 (HHV8, also known as Kaposi's sarcoma [KS]-associated herpesvirus) has been implicated as an etiologic agent for KS, an angiogenic tumor composed of endothelial, inflammatory, and spindle cells. Here, we report that transgenic mice expressing the HHV8-encoded chemokine receptor (viral G protein-coupled receptor) within hematopoietic cells develop angioproliferative lesions in multiple organs that morphologically resemble KS lesions. These lesions are characterized by a spectrum of changes ranging from erythematous maculae to vascular tumors, by the presence of spindle and inflammatory cells, and by expression of vGPCR, CD34, and vascular endothelial growth factor. We conclude that vGPCR contributes to the development of the angioproliferative lesions observed in these mice and suggest that this chemokine receptor may play a role in the pathogenesis of KS in humans.
Subject(s)
Herpesvirus 8, Human/genetics , Receptors, Chemokine/genetics , Sarcoma, Kaposi/virology , Tumor Virus Infections , Viral Proteins/genetics , Animals , CD2 Antigens/genetics , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Disease Models, Animal , Endothelial Growth Factors/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Heart Neoplasms/pathology , Hematopoietic Stem Cells/metabolism , Lymphokines/metabolism , Mice , Mice, Transgenic , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Chemokine/biosynthesis , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Kaposi/pathology , Sarcoma, Kaposi/ultrastructure , Skin Neoplasms/pathology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors , Viral Proteins/biosynthesisABSTRACT
OBJECTIVE: To assess the capacity of interleukin-4 (IL-4) and IL-10 to block polymorphonuclear neutrophil (PMN) activation in an ex vivo human model system, and to confirm their effect on neutrophil function in an animal model of arthritis. METHODS: The ex vivo phagocytic capacity of cytokine-activated human PMNs was assessed by use of assays for measuring the ingestion of heat-killed yeast and by subsequent hexose-monophosphate shunt activation using nitroblue tetrazolium reduction. The in vivo activity of IL-4 and IL-10 was measured using a rat adjuvant arthritis model in which the mycobacterial antigen concentration was titrated to modify disease intensity. RESULTS: IL-4 and IL-10 suppressed the ex vivo activation state of interferon-gamma- and tumor necrosis factor alpha-activated human neutrophils. In the rat adjuvant arthritis model, treatment with systemic murine IL-10 (mIL-10) effectively suppressed all disease parameters in rats that received the lower concentrations of mycobacteria, whereas systemic mIL-4 was effective against even the most severe disease. Both cytokines were effective in lowering the absolute PMN cell number recovered and the PMN activation state in the joint synovia. We also observed lower levels of the messenger RNA transcript for CINC protein (cytokine-induced neutrophil chemoattractant; a rat homolog for human IL-8) in the synovia. CONCLUSION: IL-10 is an effective antiarthritic agent and has a major effect on the presence and function of PMNs in the joint synovia when disease intensity is not severe. IL-4 has an inhibitory profile that is similar to that of IL-10, but is effective in modifying even the most severe disease. Both cytokines reduced the phagocytic activation of human PMNs in response to proinflammatory cytokines. These data demonstrate that IL-4 and IL-10 can exert powerful regulatory effects on neutrophil function that translate into a therapeutic response in a disease model of arthritis. Treatment with these cytokines alone or in combination may therefore be very useful in the management of patients with rheumatoid arthritis.
Subject(s)
Ankle Joint , Arthritis, Infectious/blood , Interleukin-10/pharmacology , Interleukin-4/pharmacology , Neutrophils/drug effects , Neutrophils/physiology , Tuberculosis/blood , Animals , Disease Models, Animal , Humans , Male , Mycobacterium tuberculosis , Phagocytosis/drug effects , Phagocytosis/physiology , Rats , Rats, Inbred LewABSTRACT
BACKGROUND: There have been conflicting reports of the influence of exogenous mammalian interleukin (IL)-10 on immune reactivity. These findings may reflect the pleiotropic effects of IL-10 on the functions of antigen-presenting cells and immune effector cells. The purpose of this study was to extend observations of the influence of the cytokine on organ allograft survival and to investigate its effects on the function of accessory and immune effector cells in a mouse cardiac transplant model. METHODS: C3H (H2k) recipients of heterotopic vascularized B10 (H-2b) heart allografts were treated with recombinant (r) mouse IL-10 over a wide range of doses (0.2-200 microg/day), either before the transplant (days -3, -2, -1), peri-operatively (days -1, 0, 1), or after the transplant (days 0-6). Anti-donor cytotoxic T lymphocyte activity of host spleen and graft-infiltrating cells, and circulating complement-dependent cytotoxic antibody titers were determined by isotope release assays. Mixed leukocyte reactions were used to determine the influence of IL-10 on the function of antigen-presenting cells and allogeneic responder T cells. RESULTS: Recipient pre-transplant administration of IL-10 (days -3, -2, -1) prolonged graft survival at all doses tested. Donor pretreatment with IL-10 (25 microg/day; days -3, -2, -1) was also effective, but less. A pre-transplant or perioperative course of IL-10, however, did not significantly affect the immunosuppressive action of tacrolimus given on days 0-6. If given only after the transplant, IL-10 either had no effect on graft survival or (at high dosage) accelerated rejection and prevented the immunosuppressive effect of cyclosporine. Pretransplant treatment of graft recipients with IL-10 reduced splenic anti-donor cytotoxic T lymphocyte activity and the incidence of graft-infiltrating CD8+ cells. There was no significant effect on circulating alloantibody titers. MLR assays revealed that preincubation of responder cells, but not stimulator spleen cells with IL-10, inhibited T cell proliferation, whereas addition of IL-10 after the start of culture modestly enhanced proliferation. Preincubation of purified T responders with IL-10 showed no inhibitory effect. CONCLUSION: The modest and opposing effects of exogenous IL-10 on organ allograft survival are dependent on timing and dosage. Recipient pretreatment prolongs graft survival. This finding, together with the MLR results, suggest that IL-10 inhibits the function of host immune accessory cells and that the direct pathway of alloantigen presentation may be less susceptible to inhibition by IL-10.
Subject(s)
Graft Survival/drug effects , Heart Transplantation , Interleukin-10/pharmacology , Animals , Antibodies/immunology , Graft Rejection , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology , Time Factors , Transplantation, HomologousABSTRACT
This report describes the development and the biology of Sch 55700, a humanized monoclonal antibody to human IL-5 (hIL-5). Sch 55700 was synthesized using CDR (complementarity determining regions) grafting technology by incorporating the antigen recognition sites for hIL-5 onto consensus regions of a human IgG4 framework. In vitro, Sch 55700 displays high affinity (Kd = 20 pmol/l) binding to hIL-5, inhibits the binding of hIL-5 to Ba/F3 cells (IC50 = 0.5 nmol/l) and blocks IL-5 mediated proliferation of human erythroleukemic TF-1 cells. In allergic mice, Sch 55700 (0.1-10 mg/kg, i.p. or i.m.) inhibits the influx of eosinophils in the lungs, demonstrates long duration of activity and the anti-inflammatory activity of this compound is additive with oral prednisolone. In allergic guinea pigs, Sch 55700 (0.03-30 mg/kg i.p.) inhibits both the pulmonary eosinophilia and airway hyperresponsiveness and at 30 mg/kg, i.p. inhibited allergic, but not histamine-induced bronchoconstriction. In allergic rabbits, Sch 55700 blocks cutaneous eosinophilia. Sch 55700 (0.1-1 mg/kg i.p.) also blocks the pulmonary eosinophilia and neutrophilia caused by tracheal injection of hIL-5 in guinea pigs. In allergic cynomolgus monkeys, a single dose of Sch 55700 (0.3 mg/kg i.v.) blocks the pulmonary eosinophilia caused by antigen challenge for up to six months. Sch 55700 is, therefore, a potent antibody against IL-5 in vitro and in a variety of species in vivo that could be used to establish the role of IL-5 in human eosinophilic diseases such as asthma.
Subject(s)
Antibodies, Monoclonal/pharmacology , Bronchial Hyperreactivity/pathology , Eosinophils/drug effects , Interleukin-5/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Binding, Competitive , Bronchial Hyperreactivity/immunology , Cell Division/drug effects , Cell Line , Cloning, Molecular , Eosinophilia/immunology , Eosinophilia/pathology , Eosinophils/immunology , Eosinophils/pathology , Humans , Immunoglobulin G/immunology , Interleukin-5/metabolism , Kinetics , Leukocyte Count , Lung/immunology , Lung/pathology , Macaca fascicularis , Mice , Mice, Inbred Strains , Neutrophils/pathology , Rabbits , Rats , Skin/immunology , Skin/pathologyABSTRACT
The CC chemokine known as 6Ckine (SLC, Exodus-2, or TCA4) has been identified as a ligand for CCR7. Mouse 6Ckine has also been shown to signal through mouse CXCR3 and share some of the activities of IFN-gamma inducible protein 10 and monokine induced by IFN-gamma. Nonetheless, human 6Ckine has not been shown to bind CXCR3 receptor or have angiostatic activity. In this study, we report that human 6Ckine does not induce a calcium flux in either human CXCR3 or mouse CXCR3 transfected cells, although it is an equally potent agonist as mouse 6Ckine and human macrophage inflammatory protein-3beta in human CCR7 transfected cells. Mouse 6Ckine (but not human 6Ckine) is capable of competing with radiolabeled IFN-gamma inducible protein 10 for human CXCR3. In addition, radiolabeled human 6Ckine does not bind to either human CXCR3 or mouse CXCR3. Together these data suggest that human CC chemokine 6Ckine is not a ligand for the human or mouse CXC chemokine receptor CXCR3.
Subject(s)
Chemokines, CC/physiology , Receptors, Chemokine/physiology , Signal Transduction/immunology , Animals , Cell Line , Chemokine CCL21 , Chemokines, CC/metabolism , Humans , Ligands , Mice , Receptors, CXCR3 , Receptors, Chemokine/metabolism , Species SpecificitySubject(s)
Graft Survival/immunology , Heart Transplantation/physiology , Interleukin-10/therapeutic use , T-Lymphocytes/immunology , Animals , Antibody-Dependent Cell Cytotoxicity , Cytotoxicity, Immunologic , Heart Transplantation/immunology , Isoantigens/immunology , Mammals , Mice , Mice, Inbred C3H , Mice, Inbred Strains , Recombinant Proteins/therapeutic use , T-Lymphocytes, Cytotoxic/immunology , Transplantation, HomologousABSTRACT
Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.
Subject(s)
Conserved Sequence , Nitric Oxide Synthase/chemistry , Amino Acid Sequence , Animals , Arginine/chemistry , Binding Sites , Biopterins/analogs & derivatives , Biopterins/metabolism , Crystallography, X-Ray , Drug Design , Enzyme Inhibitors/pharmacology , Heme/metabolism , Humans , Mice , Molecular Sequence Data , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitric Oxide Synthase Type III , Protein Structure, Secondary , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
We have identified and characterized a microbial extract-derived inhibitor of T cell CD28-dependent costimulation, NP1835-2, utilizing an in vitro system in which anti-human CD3 antibody and a human CD80-Ig fusion protein are immobilized on protein A-coated microspheres. This system is CD28-CD80-dependent, as judged by the specific ability of anti-CD80 antibody or cytotoxic T lymphocyte antigen-4-Ig to block human CD4 T cell responses. Activation of CD4 T cells in this system in presence of NP1835-2 resulted in a concentration-dependent inhibition of T cell proliferation (IC50 of 1-4 microg/ml), surface activation marker expression, and the production of many T cell cytokines, with the exception of TGFbeta. Impairment of T cell activation correlated with a blockade of cell cycle progression at G0/G1 and was only partly restored by addition of 100 U/ml IL-2. No inhibition by NP1835-2 of T cell proliferation stimulated by plate-bound anti-CD3 antibody, phorbol 12-myristate 13-acetate + A23187, or P815 cells expressing the costimulatory molecule CD58 was observed. NP1835-2 was unable to modulate anti-IgM-stimulated B cell proliferation or LPS-induced monocyte activation. Suboptimal concentrations of NP1835-2 and cyclosporin together were able to impair T cell activation in an additive fashion. NP1835-2 was also able to inhibit the primary human MLR. These data indicate that NP1835-2 may belong to a class of molecules capable of selectively impairing CD28-mediated T cell costimulation and suggest its potential usefulness in the treatment of a variety of T cell-dependent diseases. Moreover, NP1835-2 may serve as a useful probe for investigating the mechanisms involved in T cell nonresponsiveness.
Subject(s)
B7-1 Antigen/physiology , CD28 Antigens/physiology , CD4-Positive T-Lymphocytes/drug effects , Immunoconjugates , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , Abatacept , Antigens, CD , Antigens, Differentiation/pharmacology , CD4-Positive T-Lymphocytes/immunology , CTLA-4 Antigen , Cell Cycle/drug effects , Cyclosporine/pharmacology , Cytokines/biosynthesis , Humans , Receptors, Interleukin-2/biosynthesisABSTRACT
BACKGROUND: Endogenous interleukin (IL)-10 production has been associated with the lack of graft-versus-host disease (GVHD) in human recipients of MHC-disparate donor grafts. Paradoxically, we have shown that the exogenous administration of high doses (30 microg/dose) of IL-10 to murine recipients of MHC-disparate grafts accelerates GVHD lethality. METHODS: The effects of IL-10 on GVHD mediated by either CD4+ or CD8+ T cells was examined in studies involving exogenous IL-10 administration or the infusion of T cells from IL-10-deficient (-/-) donor mice. The role of interferon (IFN)-gamma on IL-10-induced GVHD acceleration was studied using IFN-gamma-deficient (-/-) donor mice or neutralizing monoclonal antibody. RESULTS: IL-10 was found to have a dose-dependent effect on the GVHD lethality mediated by either CD4+ or CD8+ T cells. High doses of exogenous IL-10 accelerated GVHD lethality. IFN-gamma release was not responsible for the IL-10 facilitation of GVHD lethality. Paradoxically, low doses of IL-10 protected mice against GVHD lethality. The GVHD protective effect of the bioavailability of small amounts of IL-10 was confirmed by demonstrating that the infusion of T cells from IL-10 -/- donors accelerated GVHD lethality. CONCLUSIONS: The results suggest that IL-10 has a dose-dependent effect on the GVHD lethality mediated by CD4+ or CD8+ T cells, such that high doses accelerate lethality, while low amounts of bioavailable IL-10 are protective.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Graft vs Host Disease/immunology , Interleukin-10/administration & dosage , Animals , Cytotoxicity, Immunologic , Dose-Response Relationship, Immunologic , Down-Regulation , Humans , Immunity, Cellular , Interferon-gamma/metabolism , Interleukin-10/physiology , Mice , Mice, Inbred C57BLABSTRACT
We have successfully developed a highly sensitive and specific assay system for human interleukin-4 (IL-4) regulated gene expression. It is based on a human Jijoye cell line with the germline epsilon transcript promoter joined to the human growth hormone (hGH) cDNA. The germline epsilon transcript promoter is responsive to IL-4 and involved in immunoglobulin heavy chain class switching. We cloned hGH complementary DNA (cDNA) as the reporter gene instead of using conventional hGH genomic DNA which failed to generate any IL-4 inducible clone in human Jijoye cells. The two IL-4 inducible cell lines with the hGH cDNA reporter show high signal/noise ratio for IL-4-mediated induction (60-90 fold). The response to IL-4 is dose-dependent with ED50 of 10 pM. As expected, there is no response to other human cytokines and growth factors, as well as mouse IL-4. The mutant hIL-4 antagonist hIL-4.Y124D inhibits the induction mediated by native hIL-4. These IL-4 inducible cell lines provide a sensitive, specific assay system to study IL-4-regulated gene expression, and in particular the regulation of the germline epsilon promoter.
Subject(s)
Biological Assay , Gene Expression Regulation/drug effects , Genes, Immunoglobulin , Genes, Reporter , Human Growth Hormone/genetics , Immunoglobulin epsilon-Chains/genetics , Interleukin-4/physiology , Promoter Regions, Genetic , Animals , Burkitt Lymphoma/pathology , DNA, Complementary/genetics , Human Growth Hormone/biosynthesis , Humans , Immunoglobulin epsilon-Chains/biosynthesis , Interleukin-4/antagonists & inhibitors , Interleukin-4/pharmacology , Mice , Recombinant Fusion Proteins/biosynthesis , Sensitivity and Specificity , Transfection , Tumor Cells, CulturedABSTRACT
Interleukin-17 (IL-17) has been previously reported to induce stromal cells to produce a number of hematopoietic and proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF). Here, we have evaluated the mechanisms responsible for the augmentation of G-CSF gene expression by IL-17, using the murine 3T3 fibroblast cell line. Treatment of 3T3 cells, but not primary bone marrow-derived macrophages or murine monocyte/macrophage cell lines, resulted in increased steady-state G-CSF mRNA levels within 2-4 h and augmented G-CSF protein production. The combination of IL-17 and LPS enhanced G-CSF expression in an additive fashion. Stability studies revealed that IL-17 stabilized G-CSF mRNA levels, with a t1/2 of 4 h, compared to a t1/2 of less than 2 h in medium or LPS-treated cells. Induction of G-CSF expression in 3T3 cells by IL-17 did not appear to require tyrosine kinase activation or de novo protein synthesis. These studies indicate that post-transcriptional mechanisms play an important role in IL-17-induced G-CSF expression in fibroblasts and suggest that IL-17 may be useful for further delineating mechanisms of G-CSF gene regulation.
Subject(s)
Cytokines/pharmacology , Gene Expression Regulation/drug effects , Granulocyte Colony-Stimulating Factor/genetics , Interleukins/pharmacology , 3T3 Cells , Animals , Cell Line , Cycloheximide/pharmacology , Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Interleukin-17 , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mitogens/pharmacology , Protein Synthesis Inhibitors/pharmacologyABSTRACT
CD28 is a T-cell costimulatory receptor which plays a pivotal role in antigen-induced T-cell response. We have developed a cell-free and scintillation-proximity assay-based screen to search for molecules that inhibit ligand binding to CD28. The assay was shown to be versatile and adaptable to automation for high-throughput screening. Using this assay, we identified an inhibitor of CD28, NP2214. The inhibitor was shown to be active in vitro by suppressing IL-2 synthesis and proliferation of peripheral blood mononuclear cells in response to CD28 costimulation. We also demonstrated the additive effects of NP2214 and cyclosporine A which act mechanistically distinctly in inhibiting costimulation-induced IL-2 synthesis.
Subject(s)
CD28 Antigens/metabolism , Immunoconjugates , Immunosuppressive Agents/pharmacology , Abatacept , Amino Acid Sequence , Antigens, CD , Antigens, Differentiation/metabolism , B7-1 Antigen/metabolism , CD28 Antigens/genetics , CTLA-4 Antigen , Cyclosporine/administration & dosage , Cyclosporine/pharmacology , Drug Synergism , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/isolation & purification , In Vitro Techniques , Interleukin-2/biosynthesis , Ligands , Lymphocyte Activation/drug effects , Molecular Sequence Data , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Interleukin-10 is an important cytokine that is involved in regulation of pro-inflammatory cytokines and T-cell responses. Interleukin-10 has been studied extensively in various preclinical and clinical models of inflammation. The most remarkable and consistently reproducible quality of IL-10 is its ability to downregulate macrophage functions. This includes inhibiting the production of pro-inflammatory cytokines such TNF-alpha, Interleukin-1, Interleukin-6 and antigen presentation by these professional antigen presenting cells. Additionally, Interleukin-10 also has effects on various other cell types of hematopoietic origin such as B-cells, neutrophils, and most importantly T-cells. Interleukin-10 has shown efficacy in several models of autoimmune disease. The present article deals with the effect of Interleukin-10 in animal models of inflammatory bowel disease and the results of phase I clinical trials in normal human volunteers and chronic active Crohn's disease patients.
Subject(s)
Crohn Disease/immunology , Crohn Disease/therapy , Interleukin-10/therapeutic use , Animals , Disease Models, Animal , Humans , Inflammatory Bowel Diseases/therapy , MiceABSTRACT
BACKGROUND: Systemic administration of cellular interleukin (IL)-10 at a dose of 100 microg/day for 1 week after transplantation accelerates mouse cardiac allograft rejection across MHC barriers. This effect is associated with enhancement of donor-specific cytotoxic T lymphocyte and alloantibody (alloAb) titers. To further evaluate the in vivo role of IL-10, we tested the influence of a neutralizing anti-IL-10 monoclonal antibody (mAb) in both normal and donor (skin) presensitized mouse organ allograft recipients. METHODS: Heart or liver transplants were performed from B10 (H2b) donors to C3H (H2k) recipients. Anti-IL-10 mAb (SXC.I) was administered intravenously in a single injection or repeated once daily injections. Cytotoxic activity of graft-infiltrating cells was determined by 51Cr-release assay. Circulating alloAb levels were quantified by complement-dependent cytotoxicity and flow cytometry. RESULTS: Survival of vascularized B10 cardiac allografts in normal recipients was prolonged significantly in the mAb-treated groups. A single injection of 1 mg of anti-IL-10 mAb immediately after heart transplantation gave a similar graft median survival time to repeated injections of lower dose mAb (0.5 mg/day for 6 days after transplantation) (Ig isotype control 11 days; single mAb injection 18 days; multiple injection 20 days). In presensitized recipients, anti-IL-10 mAb from days 0 to 6 significantly prolonged survival of both cardiac and orthotopic liver grafts. Graft median survival time was extended from 5 to 10 days and from 4 to 11 days, respectively. Prolongation of liver allograft survival in presensitized recipients was associated with suppression of circulating alloAb levels and with significant reductions in the incidence of B220+ cells in both grafts and recipient spleens. CONCLUSIONS: The data support an adverse role of anti-IL-10 in allograft rejection; it seems that by reducing alloAb responses, anti-IL-10 mAb may have potential for use as a therapeutic immunosuppressant, particularly in presensitized organ allograft recipients.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Graft Survival , Interleukin-10/physiology , Animals , Heart Transplantation/immunology , Immunization , Immunoglobulin G/blood , Immunoglobulin M/blood , Isoantibodies/blood , Liver Transplantation/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , T-Lymphocytes, Cytotoxic/immunology , Transplantation, HomologousABSTRACT
We have identified a small molecular weight compound, SCH 14988, which specifically stimulates in vitro granulocyte-colony stimulating factor (G-CSF) production from activated human peripheral blood mononuclear cells and monocytes but not other cytokines or CSFs with hematoregulatory activity. In vivo administration of SCH 14988 to mice rendered neutropenic by cyclophosphamide treatment resulted in the accelerated recovery of the peripheral neutrophil compartment. This activity correlated with increased in vivo G-CSF levels and stimulation of marrow granulopoiesis, and was comparable to that of exogenously administered recombinant human G-CSF. No alterations to other leukocyte populations in peripheral blood, spleen, or the peritoneal cavity were observed. These findings suggest that SCH 14988 may be clinically useful to enhance neutrophil granulopoiesis, as well as to study the mechanisms involved in G-CSF gene regulation.