Interferon-beta induces selective enhancement of antigen-specific T cell responses.

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.

Detection of allergen- and mitogen-induced human cytokine transcripts using a competitive polymerase chain reaction.

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.

Suppression of human inflammatory cell function by subtype-selective PDE4 inhibitors correlates with inhibition of PDE4A and PDE4B.

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.

Cyclic nucleotide phosphodiesterase (PDE) inhibitors and immunomodulation.

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.

Regulation of interleukin-13 by type 4 cyclic nucleotide phosphodiesterase (PDE) inhibitors in allergen-specific human T lymphocyte clones.

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.

Co-regulation of antigen-specific T lymphocyte responses by type I and type II cyclic AMP-dependent protein kinases (cAK).

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.

Anaphylaxis during autologous peripheral blood progenitor cell infusion.

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.

Cyclic nucleotide phosphodiesterases.

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.

Immunologic investigations of T-cell regulation of human IgE antibody secretion and allergic responses.

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.

Limiting dilution analysis of the frequency of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis.

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.

Differential efficacy of lymphocyte- and monocyte-selective pretreatment with a type 4 phosphodiesterase inhibitor on antigen-driven proliferation and cytokine gene expression.

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.

Differential regulation of human antigen-specific Th1 and Th2 lymphocyte responses by isozyme selective cyclic nucleotide phosphodiesterase inhibitors.

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.

Effects of nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors on antigen-induced cytokine gene expression in peripheral blood mononuclear cells.

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.

Regulation of antigen-induced human T-lymphocyte responses by calcineurin antagonists.

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.

Differential regulation of antigen-induced IL-4 and IL-13 generation from T lymphocytes by IFN-alpha.

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.

Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts.

Interleukin (IL)-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis. We describe the effects of IL-13 on collagen homeostasis from normal (NF) and keloid (KF) fibroblasts and compare these effects with those of IL-4 and transforming growth factor (TGF)-beta(1). Total collagen generation was up-regulated in NF after 48 h of stimulation by IL-13; in KF, IL-13 stimulated a more rapid collagen response. The kinetics and magnitude of collagen generation induced by IL-13 were equivalent to those induced by similar concentrations of IL-4 and TGF-beta(1). Collagen type I production paralleled total collagen generation from both NF and KF; however, IL-4-induced collagen type I and total collagen production from KF was more transient than that induced by either IL-13 or TGF-beta(1). Procollagen 1alpha1 gene expression was induced in KF by stimulation with IL-13 for 24 h. Moreover, IL-13 was unique among these three cytokines in its ability to induce gene expression for procollagen 3alpha1. Finally, IL-13 inhibited IL-1beta-induced matrix metalloproteinase (MMP)-1 and MMP-3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)-1 generation from NF; although similar effects were observed with IL-4, TGF-beta(1) transiently enhanced MMP-1 and MMP-3 generation without effecting TIMP-1. In KF, IL-13 and IL-4 inhibited MMP-3, whereas TGF-beta(1) enhanced MMP-3; TIMP-1 was unaffected by any of the three cytokines. These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.