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1.
J Interferon Cytokine Res ; 20(4): 383-9, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10805373

ABSTRACT

Interferon-beta (IFN-beta) inhibits mitogen-induced T cell responses, in part through downregulation of interleukin-12 (IL-12) or upregulation of IL-10. We have reexamined these findings using ragweed (RW) stimulated or tetanus toxoid (TT)-stimulated human peripheral blood mononuclear cells (PBMC) and nontransformed, antigen-specific, human Th0, Th1, and Th2 clones. IFN-beta induced concentration-dependent inhibition of phytohemagglutinin (PHA)-stimulated PBMC proliferation and enhancement of RW-stimulated or TTstimulated PBMC proliferation. Monocyte depletion of PBMC isolates resulted in concentration-dependent inhibition of RW-driven or TT-driven proliferation by IFN-beta. This response was unaltered by the addition of either exogenous recombinant human IL-12 (rHuIL-12) or saturating concentrations of anti-IL-10. Moreover, addition of exogenous rHuIL-10 to nondepleted RW-driven or TT-driven PBMC cultures did not alter the concentration-dependent enhancement of antigen-driven proliferation induced by IFN-beta. Th0, Th1, and Th2 clones stimulated in the presence of antigen and autologous, irradiated PBMC displayed concentration-dependent inhibition of proliferation in the presence of IFN-beta that was unaltered by the addition of either exogenous rHuIL-12 or a saturating concentration of anti-IL-10. Finally, whereas IFN-beta inhibited antigen-driven generation of IL-5, IL-12, IL-13, and IFN-gamma, IFN-beta enhanced generation of both IL-4 and IL-10. Thus, IFN-beta, induces a selective, IL-10-independent and IL-12-independent upregulation of antigen-specific T cell responses, supporting the role of IFN-beta as an immunomodulatory rather than an antiproliferative/immunosuppressive cytokine.


Subject(s)
Adjuvants, Immunologic/pharmacology , Epitopes, T-Lymphocyte/immunology , Interferon-beta/pharmacology , Cell Division/immunology , Clone Cells/cytology , Clone Cells/immunology , Clone Cells/metabolism , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/immunology , Mitogens/immunology
2.
J Immunol Methods ; 168(2): 167-81, 1994 Feb 10.
Article in English | MEDLINE | ID: mdl-8308292

ABSTRACT

Human cytokines, IL-4, IL-5, and IFN-gamma play an important role in the regulation of IgE synthesis and atopic diseases. In this communication, we describe the development of a quantitative assay of steady-state cytokine mRNAs (IL-4, IL-5, and IFN-gamma) from a variety of cell sources, including peripheral blood mononuclear cells (PBMCs) stimulated with either a mitogen (PHA) or ragweed pollen allergen extract, and cells from allergen-challenged inflammatory sites. Quantitative analysis of IL-5, IL-4 and IFN-gamma transcripts was achieved by a competitive reverse transcription-polymerase chain reaction (RT-PCR) technique using internal standard (IS) cRNAs in the presence of specific oligonucleotide primers. Each IS was generated from a plasmid vector containing the respective cytokine cDNA modified by insertion with an SV40-DNA fragment. Both test RNA and IS were reverse-transcribed and subjected to the 'competitive' PCR in the same tube. We first demonstrate the linearity and reproducibility of this technique; second, we apply this competitive PCR assay to analyze quantitatively the expression of IL-4, IL-5, and IFN-gamma transcripts in PBMCs before and after stimulation with PHA or crude ragweed allergen. Finally, we analyzed cells isolated from the lung lavage fluids of an atopic subject following allergen challenge, and showed a significant increase of IL-4 and IL-5 transcripts, but not IFN-gamma, in the allergen-challenged site when compared to the control. This technique of PCR quantitation provides an easy and efficient tool to study the expression of cytokine genes in allergic inflammatory diseases.


Subject(s)
Allergens/immunology , Cytokines/analysis , Phytohemagglutinins/immunology , Polymerase Chain Reaction/methods , RNA, Messenger/analysis , T-Lymphocytes/immunology , Base Sequence , Bronchial Provocation Tests , Bronchoalveolar Lavage Fluid/immunology , Cells, Cultured , Cytokines/genetics , DNA Primers , Electrophoresis, Agar Gel , Humans , Lymphocyte Activation/immunology , Molecular Sequence Data , Plant Lectins , Pollen/immunology , Reproducibility of Results , Rhinitis/immunology , Transcription, Genetic
3.
Br J Pharmacol ; 128(7): 1393-8, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10602317

ABSTRACT

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Immunosuppressive Agents/pharmacology , Monocytes/drug effects , Monocytes/enzymology , Phosphodiesterase Inhibitors/pharmacology , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , 3',5'-Cyclic-AMP Phosphodiesterases/classification , Animals , Antigens/immunology , Cyclic Nucleotide Phosphodiesterases, Type 3 , Cyclic Nucleotide Phosphodiesterases, Type 4 , Humans , Immunosuppressive Agents/classification , Inflammation/enzymology , Inflammation/immunology , Isoenzymes/antagonists & inhibitors , Isoenzymes/classification , Lipopolysaccharides/pharmacology , Lymphocyte Activation/drug effects , Mites/immunology , Monocytes/immunology , Monocytes/metabolism , Phosphodiesterase Inhibitors/classification , Substrate Specificity , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism
4.
Biochem Pharmacol ; 57(9): 965-73, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10796066

ABSTRACT

Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by the complex superfamily of cyclic nucleotide phosphodiesterase (PDE) enzymes. Recent advances in our understanding of the molecular pharmacology of these enzymes has led to their identification as biologic regulators of certain disease states and the development of isozyme-selective inhibitors as potential therapeutic agents. A large body of in vitro and preclinical data suggests the therapeutic utility of PDE4 inhibitors as potent anti-inflammatory agents. Early clinical trials with selective PDE inhibitors substantiate this approach while highlighting pharmacodynamic and toxicologic pitfalls inherent to the inhibition of specific PDE isozymes. This commentary will review our current understanding of PDE inhibitors as immunomodulatory agents.


Subject(s)
Cyclic AMP/pharmacology , Leukocytes/drug effects , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/metabolism , Adjuvants, Immunologic/pharmacology , Cyclic GMP/pharmacology , Humans , Immunosuppression Therapy , Leukocytes/immunology , Structure-Activity Relationship
5.
Biochem Pharmacol ; 53(7): 1055-60, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9174120

ABSTRACT

Interleukin-13 (IL-13) is a proinflammatory cytokine of T cell origin. Structural and functional studies suggest a key role for IL-13 in the genesis of chronic allergic inflammation; as such, its pharmacologic inhibition is of potential clinical utility. We studied the pharmacologic regulation of IL-13 expression by cyclic nucleotide phosphodiesterase (PDE) inhibitors in a panel of Amb a 1 (a major allergen of short ragweed, Ambrosia artemisiifolia)-specific T cell clones derived from a ragweed allergic, asthmatic subject. Proliferative responses of these cells were down-regulated by rolipram, a PDE4 inhibitor (% inhibitionMAX = 67%; IC50 = 20 microM). While the PDE3 inhibitor siguazodan provided no independent efficacy (IC50 > 10(-4) M), an increased efficacy of rolipram in the presence of 10(-5) M siguazodan was noted at 10(-6), 10(-5), and 10(-4) M rolipram (P < 0.03, 0.01, and 0.04, respectively). The EC50 values remained unchanged between assays using the PDE4 inhibitor with or without the PDE3 inhibitor. Both IL-13 gene expression and protein secretion into culture supernatants were down-regulated by the PDE4 inhibitor (P < or = 0.005). Once again, the use of a PDE3 inhibitor provided no independent efficacy (P > or = 0.2), and in this instance, increased efficacy of the PDE4 inhibitor with the PDE3 inhibitor was not apparent (P > or = 0.3). IL-13 production from clones with Th0, Th1, and Th2 phenotypes appeared equally sensitive to treatment with the PDE4 inhibitor. We conclude that the anti-inflammatory effects of PDE4 inhibitors may be mediated, in part, by down-regulation of IL-13.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Glycoproteins/pharmacology , Interleukin-13/biosynthesis , Lymphocytes/drug effects , Phosphodiesterase Inhibitors/pharmacology , Allergens , Clone Cells , Gene Expression , Guanidines/pharmacology , Humans , Interleukin-13/genetics , Lymphocyte Activation , Lymphocytes/immunology , Pyridazines/pharmacology , Pyrrolidinones/pharmacology , Rolipram
6.
Biochem Pharmacol ; 56(7): 871-9, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9774149

ABSTRACT

While a differential sensitivity to cyclic AMP (cAMP)-mediated signaling between Th1 and Th2 cells has been hypothesized, differential activity of downstream signaling through cAMP-dependent protein kinase (cAK) isoforms remains unexplored. We herein report the effects of type 1- and type 2-specific cAK agonists and antagonists on proliferative responses and cytokine generation from ragweed-driven peripheral blood mononuclear cells (PBMCs) and Amb a 1-specific Th1 and Th2 clones. Rp-8-Cl- and Rp-8-CPT-cAMP were utilized as single agent antagonists of cAKI and cAKII, respectively; 8-AHA-cAMP, with and without 8-PIP-cAMP, and 8-CPT-cAMP, with and without 6-Bnz-cAMP, were used as synergistic agonist pairs specific for the cAKI and cAKII, respectively. Activation of either cAKI or cAKII individually was ineffective in down-regulating proliferative responses of PBMCs or T cell clones; concentration-response curves for the Th1 and Th2 clones were identical. Moreover, inhibition of either cAKI or cAKII individually was ineffective in overcoming the down-regulatory effects of phosphodiesterase inhibition. Activation of either cAKI or cAKII individually was ineffective in down-regulating proinflammatory cytokine generation from T cell clones (interleukin-4 from Th2; interferon-gamma from Th1). However, concurrent activation of both cAKI and cAKII produced down-regulatory effects equivalent to those of the phosphodiesterase inhibitor on both proliferation and cytokine generation. These data suggest a critical role for concurrent activation of cAKI and cAKII in the functional efficacy of antigen-driven downstream signaling due to elevations of intracellular cAMP and argue against differential regulation of Th1 and Th2 responses by cAK subtypes.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/physiology , Epitopes, T-Lymphocyte/immunology , T-Lymphocyte Subsets/immunology , Allergens/immunology , Antigens, Plant , Clone Cells , Cyclic AMP-Dependent Protein Kinase Type II , Cytokines/biosynthesis , Down-Regulation/immunology , Enzyme Activation/drug effects , Enzyme Activation/immunology , Humans , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Plant Proteins/immunology , Plant Proteins/pharmacology , Pollen/immunology , Th1 Cells/enzymology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/enzymology , Th2 Cells/immunology , Th2 Cells/metabolism
7.
Bone Marrow Transplant ; 19(7): 749-52, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9156255

ABSTRACT

Anaphylaxis has been reported in subjects receiving peripheral blood precursor cell (PBPC) infusions; however the etiologic agent is unclear. Basophils from a PBPC-allergic subject were challenged with each individual component of the stem cell infusion and with recombinant human (rh)DNAse. Histamine release data were compared with those using basophils from control subjects. Histamine release assays were repeated using basophils from a control subject passively sensitized with serum IgE from the patient. Skin testing with bovine DNAse was performed using standard techniques. Basophil histamine release occurred in the patient, but not in controls, with bovine DNAse. No release could be provoked by any of the other components of the infusate; no release could be detected with rhDNAse. Sensitivity to bovine DNAse could be transferred to basophils from a control subject with the serum IgE from the patient. Marked epicutaneous skin test reactivity to bovine DNAse was evident in the patient, but not in control subjects. We conclude that systemic reactions during peripheral blood precursor cell infusions may represent true IgE-mediated anaphylaxis to bovine DNAse in the infusate. Skin testing can detect such sensitivity, and the use of rhDNAse may obviate such reactions.


Subject(s)
Anaphylaxis/etiology , Carcinoma/therapy , Hematopoietic Stem Cell Transplantation/adverse effects , Peritoneal Neoplasms/therapy , Animals , Cattle , Cell Separation/adverse effects , Female , Humans , Middle Aged , Transplantation, Autologous
13.
J Allergy Clin Immunol ; 108(5): 671-80, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11692087

ABSTRACT

Cyclic nucleotide second messengers (cAMP and cGMP) play a central role in signal transduction and regulation of physiologic responses. Their intracellular levels are controlled by the complex superfamily of cyclic nucleotide phosphodiesterase (PDE) enzymes. Continuing advances in our understanding of the molecular pharmacology of these enzymes has led to the development of selective inhibitors as therapeutic agents for disease states ranging from cancer and heart failure to depression and sexual dysfunction. Several PDE types have been identified as therapeutic targets for immune/inflammatory diseases. This article briefly reviews the available in vitro, preclinical, and clinical data supporting the potential for selective PDE inhibitors as immunomodulatory agents.


Subject(s)
Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/physiology , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/therapeutic use , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/enzymology , Clinical Trials as Topic , Cyclic AMP/physiology , Cyclic GMP/physiology , Endothelium/drug effects , Endothelium/enzymology , Granulocytes/drug effects , Granulocytes/enzymology , Humans , Lymphocytes/drug effects , Lymphocytes/enzymology , Mast Cells/drug effects , Mast Cells/enzymology , Models, Biological , Phosphodiesterase Inhibitors/therapeutic use , Phosphoric Diester Hydrolases/chemistry , Phosphoric Diester Hydrolases/classification
14.
Methods ; 13(1): 69-78, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9281470

ABSTRACT

The pathophysiology of allergic disease is multifactorial, involving an intricate network of interactions among cells, mediators, and cytokines. Substantial progress has been made in defining the role of antigen-specific T cells and cytokines in the regulation of immunoglobulin E (IgE) synthesis and the atopic diseases. The development of antigen-specific T-cell lines and clones has facilitated efforts to characterize human T-cell subsets and their cytokine repertoires. Molecular methods currently available include techniques for the quantitative analysis of cytokine gene expression and secretion from activated T cells ex vivo as well as in tissues. The availability of these newly developed techniques has become essential to the investigation of the pharmacologic regulation of T cells and cytokines both in vitro and in vivo. Future investigations will contribute to our understanding of the differential regulation of T-cell subsets and their relationships to allergic diseases, ultimately leading to a better understanding of the molecular pathogenesis of allergic diseases and the design of more effective therapeutic interventions.


Subject(s)
Cytokines/biosynthesis , Hypersensitivity/immunology , Immunoglobulin E/biosynthesis , T-Lymphocyte Subsets/immunology , Cytokines/genetics , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Regulation , Glucocorticoids/pharmacology , Humans , Hypersensitivity/therapy , Immunoglobulin E/immunology , In Situ Hybridization , Phosphodiesterase Inhibitors/pharmacology , Polymerase Chain Reaction , RNA, Messenger/analysis
15.
J Pharmacol Exp Ther ; 282(1): 505-12, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9223593

ABSTRACT

Our study explores the relative efficacy of phosphodiesterase (PDE) inhibitors on antigen-specific Th1 and Th2 clonal responses. Proliferative responses for both phenotypes were down-regulated by the PDE4 inhibitor, rolipram, but not the PDE3 inhibitor, siguazodan. The Th2 clones were more sensitive than the Th1 clones to PDE4 inhibition (P < .05 at 10 and 100 microM rolipram). The addition of 1 microM of the adenylyl cyclase activator, isoproterenol, significantly decreased both the EC50 and IC50 of rolipram in both phenotypes (P < .05). Gene expression for interleukin-4, interleukin-5, or interferon-gamma, assessed by reverse transcription-polymerase chain reaction, was down-regulated by the PDE4 inhibitor, but not the PDE3 inhibitor, in each respective clone. Cytokine protein secretion paralleled the results of reverse transcription-polymerase chain reaction for IL-4 and interferon-gamma (P < .01 for each). No differential efficacy on cytokine generation parameters between T helper phenotypes was apparent. Rolipram treatment significantly elevated intracellular cyclic AMP (adenosine 3',5'-cyclic monophosphate) in clonal T cells (P < .01 for Th1 or Th2 clones); these elevations were consistently greater in the Th2 clones (P < .05). Finally, Th1 cells showed reduced gene expression for the PDE4C isoform and a lack of gene expression for the PDE4D isoform by reverse transcription-polymerase chain reaction, compared to the Th2 cells. These data demonstrate the potent immunomodulatory efficacy of PDE4 inhibition on antigen-specific T cell clones. The enhanced sensitivity of Th2 cells to PDE4 inhibition may be due, in part, to the differential expression of PDE4 isoforms between Th1 and Th2 cells.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Isoenzymes/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Th1 Cells/drug effects , Th2 Cells/drug effects , Cyclic AMP/biosynthesis , Cytokines/biosynthesis , Cytokines/genetics , Gene Expression/drug effects , Humans , Lymphocyte Activation/drug effects , Pyrrolidinones/pharmacology , Rolipram , Th1 Cells/physiology , Th2 Cells/physiology
16.
Cell Immunol ; 108(1): 203-13, 1987 Aug.
Article in English | MEDLINE | ID: mdl-2440588

ABSTRACT

Lymphocytes were isolated from the spinal cord and draining lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) 12 days after immunization with myelin basic protein (MBP) and tetanus toxoid (TT). An average of 8.0 +/- 2.0 X 10(6) cells was obtained from the spinal cord. Of these 71.1 +/- 8.6% expressed the helper-T-cell marker W3/25 and 14.8 +/- 6.2% expressed the killer/suppressor-T-cell marker OX8. By limiting dilution analysis of cells exhibiting an antigen-specific proliferative response, the average frequencies of cells reactive to MBP and TT were 3.36 +/- 2.4 and 7.60 +/- 4.1 per 10(4), respectively. In the draining lymph nodes, the frequencies of cells reactive to MBP and TT were 2.24 +/- 1.7 and 2.69 +/- 2.5 per 10(4). At a relatively early stage of clinical EAE, MBP-reactive T cells comprise only a small minority of the cells which can be isolated from the spinal cord; lymphocytes reactive to a protein antigen irrelevant to EAE pathogenesis are present in comparable numbers. This finding suggests that most of these cells accumulate as a result of mechanisms not specific for MBP-reactive lymphocytes.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Lymphocytes/immunology , Animals , Antigens, Surface/analysis , Encephalomyelitis, Autoimmune, Experimental/pathology , Indicator Dilution Techniques , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocytes/pathology , Male , Myelin Basic Protein/pharmacology , Rats , Rats, Inbred Lew , Spinal Cord/pathology , Tetanus Toxoid/pharmacology
17.
J Allergy Clin Immunol ; 103(3 Pt 1): 451-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10069879

ABSTRACT

BACKGROUND: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. OBJECTIVE: We have examined the effects of IFN-alpha on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. METHODS: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. RESULTS: IFN-alpha induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-alpha was not significantly different (P =.6). IFN-alpha induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-alpha at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-alpha on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-alpha. IL-13 mRNA stability was unaffected by IFN-alpha, suggesting transcriptional regulation. CONCLUSION: IFN-alpha differentially regulates antigen-stimulated IL-4 and IL-13 generation.


Subject(s)
Antigens/immunology , Gene Expression Regulation/drug effects , Interferon-alpha/pharmacology , Interleukin-13/biosynthesis , Interleukin-4/biosynthesis , T-Lymphocyte Subsets/drug effects , Adult , Clone Cells/drug effects , Clone Cells/metabolism , Dactinomycin/pharmacology , Depression, Chemical , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-13/genetics , Interleukin-4/genetics , Jurkat Cells/drug effects , Jurkat Cells/immunology , Jurkat Cells/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Phytohemagglutinins/pharmacology , Plant Lectins , Pollen , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Rhinitis, Allergic, Seasonal/blood , Rhinitis, Allergic, Seasonal/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Th2 Cells/metabolism , Transcription, Genetic/drug effects
18.
Am J Respir Cell Mol Biol ; 13(6): 692-702, 1995 Dec.
Article in English | MEDLINE | ID: mdl-7576707

ABSTRACT

Cyclic nucleotide phosphodiesterase (PDE) enzymes may participate in regulation of the inflammatory response through their effects on second messengers. In the present study, we have investigated the role of nonselective and isozyme selective PDE inhibitors in altering the antigen-driven cytokine gene expression of peripheral blood mononuclear cells (PBMCs) from atopic individuals. Ragweed and tetanus toxoid were used as model antigens. The nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the selective PDE4 inhibitor, rolipram, markedly suppressed interleukin-5 (IL-5) and interferon gamma (IFN gamma) gene expression in both antigen-driven systems. Gene expression for IL-4 was unaffected by these agents in the ragweed-driven system. Message for IL-4 could not be detected in the tetanus toxoid-driven system, despite the use of a quantitative, competitive reverse transcription-polymerase chain reaction (RT-PCR) assay sensitive to less than 10 fg of target template. The PDE3 inhibitor, siguazodan, was ineffective in downregulating gene expression for the proinflammatory cytokines assayed; when used in combination with the PDE4 inhibitor, the PDE3 inhibitor provided no increase in efficacy over that seen with the PDE4 inhibitor alone. Gene expression for the A and B isoforms of the PDE4 in PBMCs was unaffected by antigen stimulation or treatment with the PDE4 inhibitor; however, differences in expression of these two isoforms were apparent when a variety of immune cell lines were studied. These data support the hypothesis that the primary anti-inflammatory target for PDE inhibition in PBMCs is the PDE4. Furthermore, the expression of various isoforms of this enzyme may differ between immune cell types. Finally, PDE4 isoform expression in PBMCs is independent of treatment with an isozyme selective inhibitor.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Cytokines/genetics , Leukocytes, Mononuclear/physiology , Phosphodiesterase Inhibitors/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/genetics , Allergens/immunology , Cell Division/immunology , Gene Expression/immunology , Humans , Isoenzymes/immunology , Phosphoric Diester Hydrolases/immunology , RNA, Messenger/analysis , Tetanus Toxoid/immunology
19.
J Allergy Clin Immunol ; 99(1 Pt 1): 28-37, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9003208

ABSTRACT

Elevations of intracellular cyclic AMP, achieved with the use of phosphodiesterase (PDE) inhibitors, cause functional downregulation of most inflammatory cells. Rolipram, an inhibitor selective for the PDE4 isozyme, can markedly downregulate antigen-driven proliferation and cytokine gene expression of unfractionated human peripheral blood mononuclear cells. However, it is unclear whether PDE4 inhibitors in a mixed-cell system exert their immunosuppressive effect on the lymphocyte or on the monocyte fraction. We have used an adherence-based protocol for separating peripheral blood mononuclear cells, isolated from atopic individuals, into lymphocyte and monocyte fractions and have selectively treated these populations with rolipram prior to reconstituting the cell cultures to their original lymphocyte/monocyte proportions. Cellular responses to both ragweed and tetanus toxoid were analyzed for both proliferation and gene expression of proinflammatory cytokines. A dose-dependent downregulation of ragweed- and tetanus toxoid-driven proliferative responses was achieved by pretreatment of lymphocytes from peripheral blood with rolipram. This downregulation was significantly greater than that achieved with pretreatment of monocytes. Pretreatment of both populations failed to show synergistic downregulation of proliferation. Lymphocyte pretreatment with rolipram also resulted in marked downregulation of gene expression for IL-4, IL-5, and interferon-gamma compared to monocyte pretreatment in both ragweed- and tetanus toxoid-driven systems. Interestingly, monocyte pretreatment in these systems resulted in significant downregulation of IL-2 gene expression compared to lymphocyte pretreatment. Flow cytometric analysis failed to show alterations in any of a panel of surface activation and signal transducing molecules by rolipram treatment with or without antigen stimulation. We conclude that, in a mixed cell system, PDE4 inhibitors downregulate antigen-driven proliferation and gene expression of proinflammatory cytokines predominantly through their effects on lymphocytes rather than monocytes.


Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases , Antigens/pharmacology , Cytokines/biosynthesis , Leukocytes, Mononuclear/drug effects , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/drug effects , Allergens/pharmacology , Cells, Cultured , Cyclic AMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 4 , Gene Expression/drug effects , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocytes/immunology , Lymphocytes/metabolism , Phosphoric Diester Hydrolases/metabolism , Plant Proteins/pharmacology , Pyrrolidinones/pharmacology , Receptors, Cell Surface/metabolism , Rolipram , Tetanus Toxoid/pharmacology
20.
J Allergy Clin Immunol ; 104(4 Pt 1): 828-35, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10518828

ABSTRACT

BACKGROUND: Cyclosporin A (CS) and tacrolimus (FK506, FK) are calcineurin antagonists used widely as T-cell immunosuppressants; however, their relative efficacy on antigen-stimulated T-cell subsets remains undefined. OBJECTIVE: We have examined the effects of CS and FK on antigen-driven proliferation and cytokine generation from human PBMCs and T-cell clones. METHODS: Proliferation was assessed by tritiated thymidine incorporation. Cytokine generation was assessed by reverse transcription-PCR and ELISA. RESULTS: Ragweed- and tetanus toxoid-driven proliferation of PBMCs was down-regulated equally by CS or FK. Gene expression for proinflammatory cytokines (IL-4, IL-5, IL-13, and IFN-gamma) assessed by reverse transcription-PCR was down-regulated in a concentration-dependent manner by either drug. Antigen-induced proliferation of ragweed-specific Th0, Th1, or Th2 clones was inhibited by either CS or FK. Cytokine gene expression and protein secretion into culture supernatants (IL-4, IL-5, IL-13, and IFN-gamma) were down-regulated in a concentration-dependent manner by either CS or FK in all relevant T-cell subsets. Interestingly, down-regulation of IL-5 protein generation from Th0 and Th2 clones was consistently less sensitive to either drug than was the effect on either IL-4 or IL-13 protein generation. CONCLUSION: CS and FK promote equivalent down-regulation of Th0, Th1, and Th2 responses; however, IL-5 generation is relatively insensitive to the immunomodulatory effects of calcineurin antagonists.


Subject(s)
Calcineurin Inhibitors , Cyclosporine/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes, Helper-Inducer/drug effects , Tacrolimus/pharmacology , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Hypersensitivity/immunology , Interferon-gamma/metabolism , Interleukins/metabolism , Signal Transduction/drug effects
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