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1.
Am J Respir Cell Mol Biol ; 20(3): 481-92, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10030847

ABSTRACT

We compared the effects of cyclosporin A (CSA) and a macrotetrolide antibiotic, dinactin, on human T-cell proliferation and cytokine production induced by stimulation of the T-cell receptor alone (monoclonal antibody [mAb] directed against CD3) or in combination with costimulatory signals (mAbs directed against CD3 and CD28). These agents were also examined in a murine model of interleukin (IL)-5-mediated pulmonary inflammation. Dinactin inhibited T-cell proliferation induced by IL-2, by mAb to CD3, and by mAbs to CD3 plus alpha-CD28 with identical dose-response curves (IC50 = 10-20 ng/ml). Dinactin inhibited cytokine production with IC50 values of 10 ng/ml for IL-4 and IL-5 and 30 or 60 ng/ml for interferon-gamma or IL-2, respectively. Unlike CSA, exogenous IL-2 did not alter the dinactin-mediated effects on T cells, and nuclear run-on and steady-state messenger RNA (mRNA) analysis showed that dinactin inhibited cytokine production through a post-transcriptional mechanism. CSA selectively blocked T-cell receptor-induced T-cell proliferation and cytokine production (IC50 = 10 ng/ml). Under costimulatory conditions, IL-5 synthesis was only minimally inhibited by high concentrations of CSA, and at CSA concentrations of less than 125 ng/ml, IL-5 was significantly increased above control values. Dinactin and CSA reduced pulmonary eosinophilia when administered within 1 d of airway antigen challenge. Of the cytokine mRNAs examined in the lungs of CSA-pretreated, antigen-challenged mice, IL-5 mRNA levels were the least reduced, paralleling the resistance of IL-5 to CSA observed in vitro and suggesting a role for CD28 in the in vivo induction of IL-5.


Subject(s)
Anti-Bacterial Agents/pharmacology , CD4-Positive T-Lymphocytes/immunology , Cyclosporine/pharmacology , Hypersensitivity/immunology , Interleukin-5/biosynthesis , Macrolides , Respiratory Hypersensitivity/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/immunology , Cytokines/biosynthesis , Dose-Response Relationship, Drug , Humans , Lymphocyte Activation , Mice , RNA, Messenger/analysis
2.
Immunopharmacol Immunotoxicol ; 13(3): 251-61, 1991.
Article in English | MEDLINE | ID: mdl-1940049

ABSTRACT

Mometasone furoate (9 alpha, 21 dichloro-11 beta, 17 alpha dihydroxy-16 alpha methyl-1,4 pregnadiene-3, 20 dione-17-[2'] furoate) was an unexpectedly potent inhibitor of the in vitro production of three inflammatory cytokines, IL-1(1), IL-6, and TNF-alpha. The potency of mometasone furoate in inhibiting cytokine production was compared to that of hydrocortisone, betamethasone, dexamethasone, and beclomethasone. IL-6 and TNF-alpha were both produced by WEHI-265.1 (murine myelomonocytic leukemia) cells following stimulation by lipopolysaccharide (LPS). Twenty-four hours after stimulation by LPS, the cell-free supernatant fluids were removed. Their cytokine content was analyzed using ELISAs specific for each cytokine. IL-1 synthesis was induced in the harvested peritoneal macrophages of BALB/c mice by incubation with LPS for twenty-four hours. The IL-1 content in the cell-free supernatant fluids was determined by the thymocyte-costimulator bioassay. Using these systems, mometasone furoate was found to be the most potent steroid tested for inhibiting the production of the three cytokines. The IC50's were 0.05 nM (IL-1), 0.15 nM (IL-6), and 0.25 nM (TNF-alpha). The inhibition of the production of proinflammatory mediators by extremely low concentrations of mometasone furoate suggests that this steroid should be highly effective in various disorders.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Interleukin-1/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , Pregnadienediols/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Administration, Topical , Animals , Glucocorticoids , Interleukin-1/biosynthesis , Interleukin-6/biosynthesis , Mice , Mometasone Furoate , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis
3.
J Immunol ; 139(11): 3604-13, 1987 Dec 01.
Article in English | MEDLINE | ID: mdl-2824610

ABSTRACT

Cross-linking of membrane immunoglobulin, the B cell receptor for antigen, activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by phospholipase C. This reaction yields two intracellular second messengers, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes an increase in cytoplasmic Ca2+. The experiments reported here demonstrate that activation of phospholipase C by membrane IgM (mIgM) involves a guanine nucleotide-dependent step. Saponin was used to permeabilize WEHI-231 B lymphoma cells and permit direct manipulation of nucleotide and Ca2+ concentrations. Very high levels of Ca2+ (greater than 100 microM) activated the phospholipase maximally without a requirement for cross-linking of mIgM. However, at much lower, physiologically relevant Ca2+ concentrations (100 to 500 nM), receptor-stimulated PtdInsP2 hydrolysis could be demonstrated. The ability of anti-IgM antibodies to activate phospholipase C in permeabilized WEHI-231 cells was greatly increased by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues (guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate), but not by guanosine diphosphate or guanosine diphosphate analogues or by a nonhydrolyzable analogue of adenosine triphosphate. This specificity for GTP analogues is consistent with the hypothesis that a GTP-binding regulatory protein analogous to those that couple receptors to adenylate cyclase is involved in the activation of phospholipase C by mIgM in WEHI-231 B lymphoma cells. In order to characterize this putative GTP-binding component, we examined the ability of pertussis toxin and cholera toxin to affect anti-IgM-stimulated inositol phosphate production. These bacterial toxins covalently modify and modulate the activity of various GTP-binding regulatory proteins and in some cell types can block receptor-stimulated PtdInsP2 breakdown. In WEHI-231 B lymphoma cells, neither toxin blocked signaling by mIgM. Thus mIgM appears to be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to both pertussis toxin and cholera toxin.


Subject(s)
B-Lymphocytes/physiology , GTP-Binding Proteins/physiology , Immunoglobulin M/immunology , Phosphatidylinositols/metabolism , Receptors, Antigen, B-Cell/immunology , Adenylate Cyclase Toxin , Animals , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , Calcium/metabolism , Cell Line , Cholera Toxin/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate) , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/metabolism , Mice , Pertussis Toxin , Thionucleotides/metabolism , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/physiology , Virulence Factors, Bordetella/pharmacology
5.
Science ; 234(4777): 743-6, 1986 Nov 07.
Article in English | MEDLINE | ID: mdl-3095921

ABSTRACT

Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, activates B lymphocytes and macrophages. Pertussis toxin, which inactivates several members of the G protein family of signaling components, including Gi and transducin, was found to inhibit the lipopolysaccharide-induced responses of the WEHI-231 B lymphoma cell line and the P388D1 macrophage cell line. These results, combined with the demonstration that lipopolysaccharide inhibits adenylate cyclase activity in P388D1 cells, strongly argues that lipopolysaccharide activation of cells is mediated by a Gi-like receptor-effector coupling protein.


Subject(s)
Adenylate Cyclase Toxin , B-Lymphocytes/physiology , GTP-Binding Proteins/physiology , Lipopolysaccharides/antagonists & inhibitors , Macrophages/physiology , Pertussis Toxin , Virulence Factors, Bordetella/pharmacology , Antibodies, Anti-Idiotypic/immunology , Cell Line , Dose-Response Relationship, Drug , Escherichia coli , Immunoglobulin M/immunology , Interleukin-1/metabolism , Lipopolysaccharides/immunology , Lymphocyte Activation/drug effects , Macrophage Activation/drug effects
6.
J Immunol ; 137(7): 2225-31, 1986 Oct 01.
Article in English | MEDLINE | ID: mdl-3489760

ABSTRACT

The growth of WEHI-231, a murine immature B lymphoma cell line, was inhibited by anti-IgM antibodies. The inhibition of proliferation, as measured by [3H]thymidine incorporation, occurred between 16 and 28 hr after addition of anti-IgM. Moreover, the growth arrest was irreversible: cells that were cultured with anti-IgM for 18 hr and then recultured without it failed to recover the ability to proliferate, even though cells treated for up to 30 hr with anti-IgM remained viable, as measured by trypan blue exclusion. Three polyclonal B cell activators obtained from bacteria, lipopolysaccharide (LPS), peptidoglycan from Staphylococcus aureus, and gliding bacterial adjuvant from Cytophaga (GBA), were able to protect WEHI-231 cells from anti-IgM-induced growth arrest. The protection was transient, ending after approximately 56 hr. This transience was shown to be due to desensitization of the cells to the bacterial products. Interestingly, pretreatment of WEHI-231 cells with any of the bacterial products desensitized the cells to all of the bacterial products. The heterologous nature of this desensitization suggests that all three of these bacterial products may act through a common signaling pathway despite their diverse chemical natures.


Subject(s)
B-Lymphocytes/pathology , Lymphoma/pathology , Adjuvants, Immunologic/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Cell Cycle , Cell Differentiation , Cell Line , Immune Tolerance , Immunoglobulin M/immunology , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Mice , Peptidoglycan/pharmacology , Polymyxin B/pharmacology , Polysaccharides, Bacterial/immunology , Receptors, Antigen, B-Cell/immunology
8.
J Immunol ; 133(4): 1976-82, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6432904

ABSTRACT

We assessed the dependency of a variety of immune responses for IL 2 in vitro by using anti-IL 2 receptor monoclonal antibodies as specific inhibitors of IL 2 function. The generation of allogeneic cytotoxic T lymphocyte (CTL) responses and maximal thymocyte proliferation to phytohemagglutinin (PHA) and IL 1 was readily susceptible to inhibition by these antibodies. Furthermore, the IL 2 receptor-positive, IL 2-responsive cell in the CTL cultures expressed killer cell activity. A greater variability in susceptibility to anti-IL 2 receptor antibody inhibition was noted for proliferation of T cells to concanavalin A, PHA, or allogeneic cells. Under certain conditions, however, each of these responses was almost completely inhibited. In most instances, the failure to block a response could be accounted for by either high levels of endogenous IL 2 production or high density of cell surface IL 2 receptors, which represent two known variables that influence the level of inhibition by these antibodies. Analysis of IL 2 receptor expression by mitogen-stimulated T cells suggested that accessory cells may play a role in the optimal expression of the IL 2 receptor. These experiments demonstrate that IL 2 is the predominant growth factor by which T lymphocytes proliferate, but do not exclude the possibility of an IL 2-independent pathway for growth.


Subject(s)
Antibodies, Monoclonal/physiology , Antibody Specificity , Immune Tolerance , Receptors, Immunologic/immunology , T-Lymphocytes/immunology , Animals , Interleukin-2/metabolism , Interleukin-2/physiology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mitogens/pharmacology , Receptors, Interleukin-2 , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism
9.
J Immunol Methods ; 67(2): 337-45, 1984 Mar 16.
Article in English | MEDLINE | ID: mdl-6608555

ABSTRACT

We have utilized a functional screen, inhibition of proliferation of mitogen activated lymphocytes, in an attempt to obtain monoclonal antibodies to soluble mediators of immune responses and to the receptors for such mediators. We have found that contamination of hybridoma cell lines with certain species of mycoplasma can interfere with such a screen. By consuming thymidine, mycoplasma can either mimic the effect of antibodies that inhibit lymphocyte proliferation or obscure the presence of antibodies that stimulate or enhance proliferation.


Subject(s)
Antibodies, Monoclonal/analysis , Hybridomas/microbiology , Lymphocyte Activation , Mycoplasma/isolation & purification , Animals , Antibodies/physiology , Binding, Competitive , Cell Line , Female , Hybridomas/immunology , Interleukin-1/administration & dosage , Interleukin-1/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Lymphocytes/microbiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Rats , Thymidine/metabolism
10.
Cell Immunol ; 80(1): 151-62, 1983 Aug.
Article in English | MEDLINE | ID: mdl-6603274

ABSTRACT

The nature of the defect(s) in the ability of UV-treated guinea pig macrophages to stimulate the proliferative response of guinea pig T cells to soluble protein antigens was investigated. T cells proliferated vigorously when cultured with peritoneal exudate cells (PEC) which had been pulsed with soluble protein antigens, but failed to proliferate when cultured with soluble antigen or with antigen-pulsed, UV-treated PEC. UV-treated macrophages were unable to secrete interleukin 1 (IL-1). Addition of IL-1 partially restored the T-cell proliferative response stimulated by antigen-pulsed, UV-treated PEC. However, IL-1 was able to restore such a response only when the PEC were pulsed with antigen before being exposed to UV. Similar results were obtained when antigen-pulsed PEC were used to stimulate T cells to secrete interleukin 2 (IL-2). These results demonstrate that UV-treated macrophages are defective both in their ability to properly process and present antigen for T-cell recognition and in their ability to secrete IL-1.


Subject(s)
Interleukin-1/biosynthesis , Lymphocyte Activation , Macrophages/immunology , T-Lymphocytes/metabolism , Animals , Guinea Pigs , Interleukin-2/biosynthesis , Lymphocyte Activation/radiation effects , Macrophages/radiation effects , Ovalbumin/immunology , Tuberculin/immunology , Ultraviolet Rays
12.
J Exp Med ; 146(3): 665-72, 1977 Sep 01.
Article in English | MEDLINE | ID: mdl-19551

ABSTRACT

Arginine was found to be completely depleted from cell-free supernates of mixed leukocyte cultures suppressed by the addition of excess macrophages. Partial reversal of macrophage-mediated suppression was accomplished by daily addition of a cocktail containing arginine and nonamino acid nutrients. Complete reversal of the suppression was accomplished by the addition of arginine and glucose to the medium and the nonadherent cells after their separation from the adherent macrophages. A marked increase in the enzyme arginase was found in macrophages that had been cultured 24 h in vitro in Eagle's minimum essential medium plus 10% fetal calf serum, in peritoneal cells activated by prior injection of thioglycollate, and in one spleen activated by a graft vs. host reaction.


Subject(s)
Arginase/biosynthesis , Immunosuppression Therapy , Lymphocytes/immunology , Macrophages/immunology , Animals , Arginine/pharmacology , Cytotoxicity Tests, Immunologic , Enzyme Induction , Graft vs Host Reaction , Immunity, Cellular , Macrophages/enzymology , Mice , Spleen/cytology , Thioglycolates
13.
Proc Soc Exp Biol Med ; 150(3): 791-5, 1975 Dec.
Article in English | MEDLINE | ID: mdl-1082143

ABSTRACT

In this report we investigated the ability of macrophage-associated OVA to stimulate purified OVA-immune T-lymphocytes to produce nonspecific helper activity for antibody production. T-cell activation required only a brief contact between the antigen-pulsed macrophages and T-lymphocytes and was maximal after a 4-hr contact. T-cell activation occurred at 37 but not at 4 degrees C, indicating that the macrophages and/or lymphocytes must be metabolically active for efficient activation. In addition, we compared the ability of antigen-pulsed syngeneic or allogeneic macrophages to activate T-cells for helper function.


Subject(s)
Antibody Formation , Antigens , Macrophages/immunology , T-Lymphocytes/immunology , Animals , Histocompatibility Antigens , Male , Mice , Mice, Inbred Strains , Ovalbumin/immunology , Spleen/immunology , Temperature
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