Potent Upregulation Research Articles

Differences in the regulation of BDNF and NGF synthesis in cultured neonatal rat astrocytes

Using a new brain-derived neurotrophic factor (BDNF) specific enzyme-immunoassay, we determined the basal cellular content of BDNF protein in neonatal rat astrocytes in primary culture, thus confirming the ability of astrocytes to synthesize BDNF in addition to nerve growth factor (NGF). We subsequently monitored the influence of different pharmacological agents: neurotransmitter receptor agonists, cytokines, and second messenger up-regulators, on the synthesis of BDNF and NGF. Marked differences in the regulation of their synthesis by the above pharmacological agents were observed in our study.The basal cellular levels of BDNF protein in cultured neonatal rat cortical and cerebellar astrocytes were 15.9±0.3 and 18.7±0.4pg BDNF/mg cell protein, respectively, and differ significantly between astrocytes from different brain regions, whereas NGF levels were the same (16.1±0.3 and 16.2±0.7pg NGF/mg cell protein, respectively). Screening different neurotransmitter systems for their influence on BDNF and NGF synthesis in cortical astrocytes revealed that dopamine (0.15mM) is a potent up-regulator of BDNF protein synthesis in astrocytes, while kainic acid (50μM) and histamine (1μM) did not raise the cellular level of BDNF protein. Dopamine had no influence on NGF synthesis, while kainic acid caused minor, and histamine marked, elevation of NGF cellular content. Tumor necrosis factor-α (30ng/ml) and interleukin-1β (10U/ml) treatments did not influence BDNF synthesis, whereas they markedly increased NGF protein cellular level. We also confirmed (using forskolin (20μM) and phorbol 12-myristate 13-acetate (TPA) (100nM)) that adenylate cyclase and protein kinase C participate in the downstream signaling responsible for the stimulation of BDNF synthesis, whereas in the regulation of NGF synthesis only the participation of protein kinase C was confirmed.Our results indicate that astrocyte-derived neurotrophins could play a role in distinct brain functions under physiological conditions and in the pathogenesis as well as possible treatment of different neurodegenerative disorders.

Effects of growth factors on temporomandibular joint disc cells

The effects of growth factors on cartilaginous tissues are well documented. An exception is the temporomandibular joint (TMJ) disc, where data for growth factor effects on proliferation and biosynthesis are very limited. The purpose of this study was to quantify proliferation of and synthesis by TMJ disc cells cultured in monolayer with either platelet derived growth factor-AB (PDGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-I (IGF), at either a low (10 ng/ml) or high (100 ng/ml) concentration. Proliferation was assessed with a DNA quantitation technique, collagen synthesis was measured via a hydroxyproline assay, and GAG synthesis was determined with a dimethylmethylene blue dye binding assay at 14 days. Overall, the most beneficial growth factor was bFGF, which was most potent in increasing proliferation and GAG synthesis, and also effective in promoting collagen synthesis. At the high concentration, bFGF resulted in 96% more cells than the control and 30 to 45% more cells than PDGF and IGF. PDGF and bFGF were the most potent upregulators of GAG synthesis, producing 2–3 times more GAG than the control. IGF had no significant effect on GAG production, although at its higher concentration it increased collagen production by 4.5 times over the control. Collagen synthesis was promoted by bFGF at its lower concentration, with levels 4.2 times higher than the control, whereas PDGF had no significant effect on collagen production. In general, higher concentrations increased proliferation, whereas lower concentrations favoured biosynthesis.

Differential and cooperative effects of TNFalpha, IL-1beta, and IFNgamma on human conjunctival epithelial cell receptor expression and chemokine release.

To gain better understanding of conjunctival epithelial cell responses to proinflammatory cytokines, the individual and combined effects of TNFalpha, IL-1beta, and IFNgamma on chemokine release (IL-8, regulated on activation normal T-cell expressed and secreted [RANTES]) and surface receptor expression (intercellular adhesion molecule [ICAM]-1, and HLA-DR, -DP, and -DQ) were examined. Conjunctival epithelial cells were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent. Recombinant cytokines (0.005-50 ng/mL) were added, alone or in various combinations, 24 hours before harvesting of supernates for ELISAs and cells for flow cytometry. TNFalpha, IL-1beta, and IFNgamma had distinctive individual and combined effects on the parameters tested. Although TNFalpha and IL-1beta had similar and synergistic effects on increasing expression of ICAM-1, IL-1beta was a more potent upregulator of the release of IL-8 than was TNFalpha. Upregulation of IL-8 was additive when IL-1beta was combined with TNFalpha. Neither TNFalpha nor IL-1beta increased expression of HLA. In contrast, IFNgamma was a potent upregulator of both surface receptors (ICAM-1 and HLA) but IFNgamma alone had no effect on mediator release (IL-8 and RANTES). Release of RANTES required two cytokine signals, with IFNgamma and TNFalpha being the most potent combination. Knowledge of the differential and combined effects of proinflammatory cytokines on conjunctival epithelial cells allows better understanding of ocular inflammation.

Enhancement of depsipeptide-mediated apoptosis of lung or esophageal cancer cells by flavopiridol: Activation of the mitochondria-dependent death-signaling pathway

Objective: Treating cancer cells with depsipeptide, a novel antitumor agent currently in a phase II clinical trial, causes potent upregulation of p21/WAF1 expression and cell arrest at G1 and G2 checkpoints. p21/WAF1 upregulation, however, impedes the ability of depsipeptide to induce significant apoptosis. This study was designed to determine whether flavopiridol, a synthetic cyclin-dependent kinase inhibitor known to inhibit p21 expression in tumor cells, could enhance depsipeptide-mediated apoptosis in cultured lung and esophageal cancer cells. Methods: Lung or esophageal cancer cells were exposed to depsipeptide, flavopiridol, or a combination of depsipeptide and flavopiridol. Cytotoxicity and apoptosis were quantitated by means of (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-based assays, respectively. Cytosolic cytochrome c levels, caspase 9 activity, mitochondrial membrane depolarization, and dependence of apoptosis on caspase 9 in treated cells were studied to determine the role of the mitochondria in mediating apoptosis induced by this drug combination. Results: Flavopiridol completely abolished depsipeptide-mediated dose-dependent upregulation of p21/WAF1 expression. Combining flavopiridol with depsipeptide resulted in a 3- to 8-fold reduction of depsipeptide inhibitory concentration of 50% values that was closely paralleled by synergistic enhancement of apoptosis (4- to 10-fold higher than levels of cell death induced by either drug alone) in all cancer cell lines. The essential role of mitochondria in mediating cell death was indicated by robust translocation of cytochrome c from the mitochondria into the cytosol, 2.5- to 5-fold activation of caspase 9, severe disruption of mitochondrial inner membrane potential, and complete inhibition of apoptosis by the selective caspase 9 inhibitor. More important, this drug combination was not toxic to primary normal epithelial cells derived from the airway or skin. Conclusion: The depsipeptide plus flavopiridol combination exhibits powerful and selective cytocidal activity against cancer but not normal cells. Apoptosis induced by this combination is mediated by the mitochondria-dependent death pathway.J Thorac Cardiovasc Surg 2003;125:988-91

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: transcriptional regulation by TNFα

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFα, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFα may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Activation of the MEF2 transcription factor in skeletal muscles from myotonic mice

Becker syndrome, a recessive nondystrophic myotonia caused by mutations in the chloride channel 1 gene (CLCN1), is characterized by delayed muscle relaxation after contraction. The ADR (arrested development of righting response) mouse is an animal model for Becker syndrome. Skeletal muscles from ADR myotonic animals show an increased number of oxidative fibers with a lack of glycolytic fibers as well as signs of muscle hypertrophy. Through breeding ADR myotonic mice with mice harboring a MEF2-dependent reporter gene, we found that the transcriptional activity of MEF2 was dramatically enhanced in myotonic muscles. Post-translational induction of MEF2 transcriptional activity correlated with the activation of p38 MAPK and did not affect MEF2 DNA-binding affinity. Expression of class II histone deacetylases (HDACs), which repress MEF2-dependent gene expression, was significantly reduced in skeletal muscles from myotonic mice. These findings suggest that the combined effects of class II HDAC deficiency and p38 MAPK activation lead to potent upregulation of MEF2 transcriptional activity, which contributes to the long-term changes in gene expression and fiber-type transformation observed in myotonic skeletal muscles. These findings provide new molecular targets for potential treatment of congenital myotonia.

Activation of the MEF2 transcription factor in skeletal muscles from myotonic mice

Becker syndrome, a recessive nondystrophic myotonia caused by mutations in the chloride channel 1 gene (CLCN1), is characterized by delayed muscle relaxation after contraction. The ADR (arrested development of righting response) mouse is an animal model for Becker syndrome. Skeletal muscles from ADR myotonic animals show an increased number of oxidative fibers with a lack of glycolytic fibers as well as signs of muscle hypertrophy. Through breeding ADR myotonic mice with mice harboring a MEF2-dependent reporter gene, we found that the transcriptional activity of MEF2 was dramatically enhanced in myotonic muscles. Post-translational induction of MEF2 transcriptional activity correlated with the activation of p38 MAPK and did not affect MEF2 DNA-binding affinity. Expression of class II histone deacetylases (HDACs), which repress MEF2-dependent gene expression, was significantly reduced in skeletal muscles from myotonic mice. These findings suggest that the combined effects of class II HDAC deficiency and p38 MAPK activation lead to potent upregulation of MEF2 transcriptional activity, which contributes to the long-term changes in gene expression and fiber-type transformation observed in myotonic skeletal muscles. These findings provide new molecular targets for potential treatment of congenital myotonia.

Effects of signaling molecules, protein phosphatase inhibitors and blast pathogen (Magnaporthe grisea) on the mRNA level of a rice (Oryza sativa L.) phospholipid hydroperoxide glutathione peroxidase (OsPHGPX) gene in seedling leaves

With a specific focus on rice self-defense response(s), the effects of global signaling molecules, jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and ethylene (using the ethylene generator, ethephon), and protein phosphatase (PP) inhibitors, cantharidin and endothall on expression of a rice phospholipid hydroperoxide glutathione peroxidase (OsPHGPX) gene in rice seedling leaves were investigated. We provide first evidence for a potent up-regulation of the OsPHGPX mRNA accumulation by these signaling molecules and PP inhibitors that strongly suggest its potential role in defense/stress. The OsPHGPX gene also showed a weak constitutive expression and responsiveness to cut. These inductions were influenced by light signal(s), and did not show a requirement for de novo synthesized protein factor(s). A potential interaction amongst these signaling molecules, especially JA, SA, ABA and kinetin, in modulating the OsPHGPX expression was found. The blast pathogen, Magnaporthe grisea also elicited the accumulation of OsPHGPX mRNA in leaves. This is a first systematic report in rice (and in plants) demonstrating the inducible nature (and expression) of the OsPHGPX gene by a variety of defense/stress-related stimuli, and modulation by the PPs of the kinase-signaling cascade(s).

Control of target cell survival in thyroid autoimmunity by T helper cytokines via regulation of apoptotic proteins

After autoimmune inflammation, interactions between CD95 and its ligand (CD95L) mediate thyrocyte destruction in Hashimoto's thyroiditis (HT). Conversely, thyroid autoimmune processes that lead to Graves' disease (GD) result in autoantibody-mediated thyrotropin receptor stimulation without thyrocyte depletion. We found that GD thyrocytes expressed CD95 and CD95L in a similar manner to HT thyrocytes, but did not undergo CD95-induced apoptosis either in vivo or in vitro. This pattern was due to the differential production of TH1 and TH2 cytokines. Interferon gamma promoted caspase up-regulation and CD95-induced apoptosis in HT thyrocytes, whereas interleukin 4 and interleukin 10 protected GD thyrocytes by potent up-regulation of cFLIP and Bcl-xL, which prevented CD95-induced apoptosis in sensitized thyrocytes. Thus, modulation of apoptosis-related proteins by TH1 and TH2 cytokines controls thyrocyte survival in thyroid autoimmunity.

Interleukin-8 fails to induce human immunodeficiency virus-1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines.

This study addresses the role of interleukin (IL)-8, a CXC-chemokine, the level of which is reported to be raised in the peripheral circulation of human immunodeficiency virus-1 (HIV-1)-infected individuals, during the induction of HIV-1 expression from latency and during cytokine-mediated HIV-1 up-regulation. IL-8 at the higher concentrations tested (> or = 100 ng/ml) was unable to induce HIV-1 expression in the chronically infected promonocytic U1 cell line, as measured by p24 antigen enzyme-linked immunosorbent assay (ELISA), whereas at lower concentrations of 1 and 10 ng/ml, constitutive HIV-1 expression was only marginally reduced. HIV-1 replication in acutely infected U937 cells was also significantly reduced by IL-8. The potent up-regulation of HIV-1 expression in U1 cells by tumour necrosis factor-alpha (TNF-alpha) remained unaffected by the addition of IL-8. HIV-1 induction by IL-1beta, IL-6 and TNF-beta, cytokines grouped here as intermediate HIV-1 inducers, was suppressed by IL-8 at concentrations of 1 and 10 ng/ml. However, IL-8 at 100 ng/ml did not significantly alter the effect of IL-1beta, synergized with IL-6 in enhancing, and marginally suppressed TNF-beta-induced HIV-1 expression. IL-8 suppressed granulocyte-macrophage colony-stimulating factor (GM-CSF) and enhanced interferon-gamma (IFN-gamma)-induced HIV-1 expression in a dose-dependent manner. Pretreatment of U1 cells with IL-8 did not alter the IL-8-mediated effects on cytokine-induced HIV-1 expression, suggesting that this chemokine exerts its effect at the time of HIV-1 induction or at a postinduction stage. Furthermore, IL-8 was itself induced by cytokines that up-regulate HIV-1 expression in U1 cells and the levels produced correlated directly with the levels of p24 antigen produced, suggesting common pathways for cytokine induction of both HIV-1 and IL-8. These results show that IL-8, typically a non-inducer, can differentially modulate HIV-1 expression in U1 cells and that this is dependent on the inducing cytokine and on the concentration of IL-8.

Interleukin-9–Induced Expression of M-Ras/R-Ras3 Oncogene in T-Helper Clones

In an attempt to gain insight into the molecular mechanisms involved in interleukin-9 (IL-9) activities, representational difference analysis (RDA) was used to identify messages that are induced by IL-9 in a murine T-helper–cell clone. One of the isolated genes encodes for the newly described M-Ras or R-Ras3, which is part of the Ras gene superfamily. M-Ras expression was found to be induced by IL-9 but not IL-2 or IL-4 in various murine T-helper–cell clones, and this induction seems to be dependent on the JAK/STAT pathway. Contrasting with the potent upregulation of M-Ras expression, M-Ras was not activated by IL-9 at the level of guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding. However, IL-3 increased GTP binding to M-Ras, suggesting that M-Ras induction might represent a new mechanism of cooperativity between cytokines such as IL-3 and IL-9. Constitutively activated M-Ras mutants induced activation of Elk transcription factor by triggering the MAP kinase pathway and allowed for IL-3–independent proliferation of BaF3 cells. Taken together, these results show that cytokines such as IL-9 can regulate the expression of a member of the RAS family possibly involved in growth-factor signal transduction.

Interleukin-9–Induced Expression of M-Ras/R-Ras3 Oncogene in T-Helper Clones

AbstractIn an attempt to gain insight into the molecular mechanisms involved in interleukin-9 (IL-9) activities, representational difference analysis (RDA) was used to identify messages that are induced by IL-9 in a murine T-helper–cell clone. One of the isolated genes encodes for the newly described M-Ras or R-Ras3, which is part of the Ras gene superfamily. M-Ras expression was found to be induced by IL-9 but not IL-2 or IL-4 in various murine T-helper–cell clones, and this induction seems to be dependent on the JAK/STAT pathway. Contrasting with the potent upregulation of M-Ras expression, M-Ras was not activated by IL-9 at the level of guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding. However, IL-3 increased GTP binding to M-Ras, suggesting that M-Ras induction might represent a new mechanism of cooperativity between cytokines such as IL-3 and IL-9. Constitutively activated M-Ras mutants induced activation of Elk transcription factor by triggering the MAP kinase pathway and allowed for IL-3–independent proliferation of BaF3 cells. Taken together, these results show that cytokines such as IL-9 can regulate the expression of a member of the RAS family possibly involved in growth-factor signal transduction.

Development of Phagocytic Function of Cultured Human Monocytes Is Regulated by Cell Surface IL-10

Monocytes differentiatingin vitrointo macrophages increase their capacity to ingest particles via FcγR and CR3. Because human recombinant IL-10 is a potent up-regulator of phagocytosis in human monocytes, we investigated whether spontaneously produced IL-10 could be a signal for the modulation of phagocytosis by cultured monocytes. We show here that culture of monocytes in the presence of anti-IL-10 mAb completely abolished up-regulation of phagocytosis of both EIgG and EIgMC3bi, suggesting a role for spontaneously produced IL-10 in the modulation of phagocytosis by cultured human monocytes. The inhibition exerted by anti-IL-10 mAb on the development of FcγR-mediated ingestion was dependent on the concomitant inhibition of FcγRIII induction in cultured cells. On the other hand, a similar down-regulation of CR3 expression was not involved in the inhibitory effect exerted by anti-IL-10 mAb on the development of CR3-mediated ingestion. Monocytes secreted detectable levels of IL-10 when cultured in medium but the concentrations of IL-10 in the supernatants decreased with length of time in culture, the decrease being completely reversed by anti-IL-10 mAb. In addition, we showed that monocytes expressed immunoreactive IL-10 on their surface and this expression increased during differentiation into macrophages. Whether this IL-10 was bound to specific membrane receptors or it was an integral membrane protein remains to be determined; however, this latter possibility is consistent with our observations that IL-10 did not elute with acid treatment and exogenous IL-10 did not increase surface staining of monocytes. Our data indicate that human mononuclear phagocytes express IL-10 on their membrane and suggest that this cytokine may represent an autocrine signal for the increased phagocytic function observed during differentiation of monocytes into macrophages.

Constitutive activation of delayed-rectifier potassium channels by a src family tyrosine kinase in Schwann cells.

In the nervous system, Src family tyrosine kinases are thought to be involved in cell growth, migration, differentiation, apoptosis, as well as in myelination and synaptic plasticity. Emerging evidence indicates that K+ channels are crucial targets of Src tyrosine kinases. However, most of the data accumulated so far refer to heterologous expression, and native K+-channel substrates of Src or Fyn in neurons and glia remain to be elucidated. The present study shows that a Src family tyrosine kinase constitutively activates delayed-rectifier K+ channels (IK) in mouse Schwann cells (SCs). IK currents are markedly downregulated upon exposure of cells to the tyrosine kinase inhibitors herbimycin A and genistein, while a potent upregulation of IK is observed when recombinant Fyn kinase is introduced through the patch pipette. The Kv1.5 and Kv2.1 K+-channel alpha subunits are constitutively tyrosine phosphorylated and physically associate with Fyn both in cultured SCs and in the sciatic nerve in vivo. Kv2.1- channel subunits are found to interact with the Fyn SH2 domain. Inhibition of Schwann cell proliferation by herbimycin A and by K+-channel blockers suggests that the functional linkage between Src tyrosine kinases and IK channels could be important for Schwann cell proliferation and the onset of myelination.

Hypocholesterolemic effects of the LDL receptor gene transcriptional upregulator CP-230821.

CP-230821 is a novel, potent LDL receptor gene transcriptional upregulator which decreases total plasma cholesterol level. Interestingly, this plasma LDL decrease does not alter hepatic lipid contents. A series of experiments was undertaken to study the molecular biology of this phenomenon. Twelve hours after CP-230821 treatment, the transcriptional activity and mRNA level of the LDL receptor gene in HepG2 cells were increased by 264% and 426%, respectively. Although treatment with the HMG-CoA reductase inhibitor compactin also increased LDL receptor gene transcription and mRNA, CP-230821 did not increase the level of HMG-CoA reductase gene transcription or mRNA. These results indicate that LDL receptor gene activity may play an important role in the decrease of plasma LDL level. These results further suggest that the LDL receptor gene and the HMG-CoA reductase gene are not strictly coordinately controlled.

Synthesis of 4α-(2-propenyl)-5,6-secocholestan-3α-ol, a Novel B-ring Seco Analog of the Hypocholesterolemic Agent 4α-(2-propenyl)-5α-cholestan-3α-ol

4α-(2-Propenyl)-5α-cholestan-3α-ol (LY295427) was previously identified from a CHO cell-based assay to be a potent LDL receptor up-regulator and had demonstrated to be an effective agent in lowering plasma cholesterol levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3α-hydroxy-bearing A-ring on the activity, 4α-(2-propenyl)-5,6-secocholestan-3α-ol ( 11), a B-ring seco analog of LY295427, is thus synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL receptor/luciferase assay at concentrations up to 20 μg/mL. The result underlines the importance of maintaining the A-B-C-D ring rigidity of the 3α-sterols in terms of binding to the putative oxysterol receptor.

The TH1 lymphokine interferon-gamma is a potent upregulator of dendritic cells with phagocytic capacity in GM-CSF supplemented bone marrow cultures.

Myeloid dendritic cells (DC), macrophages and granulocytes are descendants of a hematopoietic progenitor cell that originates in the bone marrow1. Thus, bone marrow derived cells distributed in tissue culture in the presence of GM-CSF give rise to the three leukocyte populations which under various in vitro culture conditions proceed in differentiation and phenotypic maturation2–7.KeywordsDendritic CellCutaneous LeishmaniasisMouse Bone MarrowBone Marrow CulturePhagocytic CapacityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Receptor binding specificity and pulmonary gene expression of the neutrophil-activating peptide ENA-78.

Neutrophil-activating peptide ENA-78 is a novel chemotactic cytokine isolated from a human type II pulmonary epithelial cell line. It is a member of the chemokine family of proinflammatory polypeptides and exhibits structural homology to interleukin-8 (IL-8) and GROalpha. The immunohistochemical identification of ENA-78 in pulmonary alveolar leukocytes of bovine pneumonic lungs supports a role for ENA-78 in the pathogenesis of pulmonary inflammation. Although ENA-78 is able to stimulate polymorphonuclear neutrophils (PMN), neither its binding specificities nor its expression in human pulmonary disease states have been determined. 125I-labeled ENA-78 binds with high affinity to human PMN. Its actions on PMN appear to be mediated by the IL-8 type B receptor, to which it binds with a K(d) of 2.2 nM. Human IL-8, GROalpha, and murine KC compete with high affinity for 125I-ENA-78 binding to the human IL-8 type B receptor. In contrast, 125I-ENA-78 does not bind to the IL-8 type A receptor nor does it compete significantly for 125I-IL-8 binding to this same receptor. ENA-78 is a potent upregulator of Mac-1 cell surface expression. In addition, ENA-78 mRNA is detected in cystic fibrosis lung but is not detected in normal donor lung. Thus, ENA-78 mRNA levels appear to be increased in human pulmonary inflammation and its stimulatory activities on PMN appear to be a function mediated primarily by the IL-8 type B receptor.

IL-10 up-regulates human monocyte phagocytosis in the presence of IL-4 and IFN-γ

Interleukin-10 (IL-10), a cytokine produced by type 2 helper T (Th2) cells, inhibits the microbicidal effector function of interferon-gamma (IFN-gamma)-activated macrophages. However, recent observations indicate that IL-10, like IFN-gamma, increases Fc gamma RI expression and Fc gamma R-mediated cytotoxic activity on human monocytes, suggesting that this cytokine cannot be classified purely as a monocyte deactivator. The present study found that incubation for 40 h of human monocytes or monocyte-derived macrophages in the presence of IL-10 caused a significant enhancement of their capacity to ingest particles coated with immunoglobulin G (Fc gamma R-mediated ingestion) or with C3b/C3bi fragments of the complement system (CR1/CR3-mediated ingestion). The number of phagocytosing cells (% phagocytosis) and the number of ingested particles per cell (phagocytic index) were both significantly higher after 40-h incubation of monocytes with IL-10 concentrations > or = 1 U/ml. This up-regulating activity on phagocytosis was completely reversed by anti-IL-10 monoclonal antibody (mAb). As previously reported, IL-10 stimulated Fc gamma RI expression on monocytes but did not induce the expression of Fc gamma RII, Fc gamma RIII, CR1, and CR3. IFN-gamma, like IL-10, up-regulated only Fc gamma RI expression but significantly reduced both Fc gamma R- and CR-mediated ingestion. IL-10 almost completely reversed the IFN-gamma-induced inhibition of both Fc gamma R- and CR-mediated phagocytosis, without concomitant changes in membrane expression of phagocytic receptors. Exposure of monocytes to IL-4 reduced the membrane expression of all three Fc gamma Rs and also inhibited Fc gamma R-mediated ingestion. On the other hand, IL-4 up-regulated both CR3 expression and CR-mediated ingestion on cultured monocytes. IL-10 not only neutralized the down-regulatory effect of IL-4 on Fc gamma R expression but also completely reversed the IL-4-induced suppression of Fc gamma R-mediated phagocytosis. Exposure of monocytes to a combination of IL-10 and IL-4 resulted in a synergistic effect on CR-mediated ingestion, even though no additive effects were observed on CR membrane expression. Finally, culture of monocytes in medium containing anti-IL-10 mAb significantly reduced their capacity to ingest IgG- or C3b/C3bi-coated particles, suggesting a role for endogenously produced IL-10 in the modulation of phagocytosis by human monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Upregulated expression of IL-4 receptors and increased levels of IL-4 in rheumatoid arthritis patients

The level of IL-4R expression on peripheral lymphocyte subsets from rheumatoid arthritis (RA) patients and controls was studied by flow cytometric analysis of the binding of phycoerythrin-labelled IL-4 (PE-IL4). In normal lymphocytes, IL-4R is mainly expressed on CD19 + cells, although it was also seen, at lower levels, on CD3 +, CD4 + and CD8 + cells. In RA patients, a significantly increased spontaneous expression of IL-4R was observed, compared with controls, in the CD3 +, CD4 + and CD19 + cell subsets. No significant differences in IL-4R expression were found between patients receiving steroids and those who were not, suggesting that steroids are not involved in upregulating IL-4R levels in vivo. Because IL-4 is a potent upregulator of IL-4R, we considered the possibility that incremented levels of circulating IL-4 in RA accounted for the high surface expression of IL-4R. By ELISA, we found abnormally high levels of immunoreactive IL-4 in 35.13% of patient serum samples, while it was undetectable in control sera. In addition, we examined IL-4 mRNA expression by polymerase chain reaction (PCR) in the PBMC of patients and controls. IL-4 PCR products were observed in four out of 10 patients studied but in none of the controls. No correlation was observed between the seric concentrations of IL-4 and IL-4R, indicating that activator factors other than IL-4 contribute to the upregulation of IL-4R expression in RA. Since the patients' sedimentation rate and CRP values did not correlate with the concentration of circulating IL-4, we conclude that this lymphokine does not contribute to the deleterious effect of the disease. Rather, due to its antiinflammatory properties, the overproduction of IL-4 in RA may be a compensatory mechanism neutralizing the harmful effect of activated macrophages.