Nortriptyline Modulates the Migration of Peripheral Blood Lymphocytes and Monocytes in Patients with Chronic Obstructive Pulmonary Disease.

In the present study, the effect of nortriptyline (1 and 10 µM), budesonide (10 nM) and their combination on the migration of peripheral blood lymphocytes and monocytes from patients with chronic obstructive pulmonary disease (COPD) towards chemokines CCL5 and CXCL10 was evaluated by flow cytometry. Nortriptyline (10 µM), both alone and in combination with budesonide, inhibited the migration of T helper cells, cytotoxic T lymphocytes, NK cells and B lymphocytes towards CCL5 and CXCL10, as well as enhanced monocyte migration towards these chemokines. The combination of nortriptyline (1 µM) and budesonide suppressed the chemotaxis of lymphocyte subpopulations towards CXCL10, but not towards CCL5, more effectively than budesonide alone. The combination of nortriptyline (10 µM) and budesonide inhibited the migration of lymphocyte subpopulations towards CCL5 and CXCL10 and activated monocyte chemotaxis towards both chemokines more effectively than budesonide alone. The results of this study demonstrate the ability of nortriptyline alone to modulate the migration of peripheral blood lymphocytes and monocytes from patients with COPD and to potentiate the effects of budesonide.

[The effect of glucocorticoids in combination with azithromycin or theophylline on cytokine production by NK and NKT-like blood cells of patients with chronic obstructive pulmonary disease]. / Vliianie gliukokortikosteroidov pri ikh sovmestnom primenenii s azitromitsinom ili teofillinom na produktsiiu tsitokinov NK i NKT-podobnymi kletkami krovi patsientov s khronicheskoi obstruktivnoi bolezn'iu legkikh.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced sensitivity of cells to the anti-inflammatory effects of glucocorticoids (GCs). Azithromycin and a low dose theophylline have a significant impact on molecular mechanisms leading to corticosteroid resistance. The aim of this study was to evaluate the ability of azithromycin and theophylline to enhance the anti-inflammatory effects of GCs on the production of cytokines by NK and NKT-like blood cells of COPD patients. Whole blood cells from COPD patients (n=21) were incubated in the presence of budesonide (10 nM), azithromycin (10 µg/mL), theophylline (1 µM), or their combinations and stimulated with phorbol myristate acetate (50 ng/mL). Intracellular production of proinflammatory cytokines in NK (CD3-CD56+) and NKT-like (CD3+CD56+) blood cells was analyzed by flow cytometry. Budesonide reduced synthesis of interleukin 4 (IL-4), CXCL8, tumor necrosis factor α (TNFα) by NK and NKT-like cells, as well as production of interferon γ (IFNγ) by NK cells. Azithromycin suppressed production of IL-4 and CXCL8 by NK and NKT-like cells, and theophylline inhibited IL-4 synthesis by these lymphocytes. The combination of azithromycin and budesonide had a more pronounced inhibitory effect on the production of IL-4 and CXCL8 by NK and NKT-like cells than the effect of these drugs alone. The combination of theophylline and budesonide suppressed synthesis of IL-4 and CXCL8 by NK and NKT-like cells, as well as the production of TNFα and IFNγ by NK cells stronger than budesonide alone. The obtained results demonstrate the benefits for the combined use of GCs with theophylline at a low dose or azithromycin to suppress the inflammatory process in patients with COPD.

Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells.

Defensins, a family of small, cationic, antimicrobial peptides, are found in mammals, insects and plants. alpha-defensins are stored in granules of neutrophils and released upon activation by exocytosis. It was shown here that human neutrophil peptide (HNP), at concentrations of 10(-8) -10(-9) M, up-regulated the expression of TNF-alpha and IL-1 beta in monocytes activated with Staphylococcus aureus or PMA, while expression of IL-10 mRNA was down-regulated and production of IL-8 was not affected. HNP alone was unable to induce TNF-alpha or IL-1 beta expression in resting monocytes. At concentrations of 10(-4) -10(-5)M, HNP was cytotoxic for monocytes in serum-free medium. The cytotoxicity was abrogated in the presence of serum, while a cytokine-modulating effect of HNP was observed in the presence of serum and in whole blood, suggesting that this mechanism may function in vivo. Similarly, serum did not abrogate bactericidal activity of HNP. It was also demonstrated herein that HNP at 10 (-8) -10(-9) M, attenuated the inhibitory action of dexamethasone on TNF-alpha production. In parallel to monocyte studies, we have showed that HNP at concentrations ranging from 10(-9)M to 10(-6)M caused about 5-fold suppression of VCAM-1 expression in TNF-alpha-activated human umbilical vein endothelial cells, while the ICAM-1 expression was not affected. Our findings suggest that neutrophil defensins have the potential to modulate the inflammatory responses through regulation of cytokine production and adhesion molecule expression.

Expression of IL-8 gene in human monocytes and lymphocytes: differential regulation by TNF and IL-1.

TNF-alpha and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-alpha- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF-alpha with the induction of IL-8 mRNA or protein, nor require endogenous TNF-alpha for IL-8 expression. In contrast, in the presence of exogenous IL-1alpha and IL-1beta a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1alpha and IL-1beta were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-alpha, similar to exogenous IL-1alpha and IL-1beta. As was seen with MO, antibodies to TNF-alpha, IL-1alpha and IL-1beta did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF-alpha and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli.

Differential action of cycloheximide and activation stimuli on transcription of tumor necrosis factor-alpha, IL-1 beta, IL-8, and P53 genes in human monocytes.

In the present study we have analyzed superinduction of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription by cycloheximide (Chx) in human blood monocytes isolated by continuous Percoll gradient and activated in vitro. In the same monocyte cultures, we have compared the rate of gene transcription of TNF-alpha, IL-1 beta, IL-8, and the P53-antioncogene under the influence of plastic adherence, Staphylococcus aureus Cowan 1 (SAC), and Chx added at different times of monocyte culture. It was shown that the cytokine genes have low or negligible transcriptional activity in freshly isolated monocytes, whereas P53 gene transcription was constant in freshly isolated and in vitro-stimulated cells. Transcription of the IL-1 beta and IL-8 genes was induced by adherence and was not more enhanced by SAC. Transcription of the TNF-alpha gene was not induced by adherence. Chx added at the beginning of the monocyte culture did not block TNF-alpha or IL-1 beta gene transcription. IL-8 gene transcription, however, was abrogated by Chx. Addition of SAC to monocyte culture containing Chx caused significant enhancement of TNF-alpha gene transcription. Addition of Chx after 2.5 or 4 h of SAC activation caused "superinduction" of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription. The data imply that TNF-alpha gene transcription in activated human monocytes might be regulated by both positive and negative regulatory factors that differ in their stability and protein synthesis dependence. In addition, results demonstrate that TNF-alpha, IL-1 beta, IL-8, and p53 genes in human monocytes are differently regulated.

The effect of biotinylation on the antigenic specificity of anti-defensin monoclonal antibodies.

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies. The effects of NHS-biotinylation were in part mimicked by 2,4-dinitrofluorobenzene, another agent that modified primary amine groups of proteins, suggesting that this modification contributed to the change in antibody specificity. When a high degree of antigenic specificity against peptide immunogens is required, biotinylation on the glycan moiety of Mabs may be preferable.

[Differences in anti-oncogene p53 expression in human monocytes and lymphocytes in vitro]. / Razvitiia v ékspressii "antionkogena" p53 v monotsitakh i limfotsitakh cheloveka in vitro.

The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.

[Effects of human defensin HNP-1 on the production of tumor necrosis factor-alpha by human blood monocytes in vitro]. / Vliianie defenzina HNP-1 neitrofilov cheloveka na produktsiiu faktora nekroza opukholei-al'fa monotsitami krovi cheloveka in vitro.

The influence of human defensin HNP-1 on the synthesis of tumor necrosis factor-alpha (TNF-alpha) by human blood monocytes was studied. It was shown that TNF-alpha production by human monocytes activated by SAC or PMA was augmented in the presence of HNP-1 concentrations of 10(-8)-10(-9) M. HNP-1 alone induced no synthesis of TNF-alpha. High concentration of HNP-1 (10(-4) M) was cytotoxic for human monocytes.

The difference in p53 antioncogene transcription in human monocytes and lymphocytes.

The p53 gene is associated with malignant transformation as well as 'antioncogene' activity. In this report expression of p53 in resting and activated human blood monocytes and lymphocytes was studied. It is shown that human monocytes freshly isolated by continuous Percoll-gradient centrifugation contain detectable levels of p53 mRNA. Stimulation of monocytes by the potent activation inducer Staphylococcus aureus Cowan I (SAC) for 3-5 h caused the disappearance of p53 mRNA. In contrast, induction of a high level of tumor necrosis factor alpha mRNA was detected. The addition of cycloheximide did not increase the p53 mRNA content in stimulated monocytes, and decreased the mRNA level in resting cells. p53 mRNA was absent in freshly isolated lymphocytes and in resting cells cultured for 20 h. Activation of lymphocytes by phytohemagglutinin caused accumulation of p53 mRNA. We suggest that p53 gene regulation and functions might be different in human monocytes and lymphocytes.

[The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]. / O mekhanizme protivovirusnoi aktivnosti antivirusnogo faktora neinterferonovoi prirody.

Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.

[Synthesis of tumor necrosis factor-alpha (TNF-alpha) by human monocytes in vitro]. / Sintez faktora nekroza opukholei-alfa(FNO-alfa) monotsitami cheloveka in vitro.

The presence of TNF-alpha mRNA and protein in circulating human blood monocytes isolated by continuous Percoll gradient fractionation was studied. The technique of RNA isolation from the blood samples was used to study TNF-alpha mRNA expression. It was shown that human blood monocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein.

Tumor necrosis factor alpha induction in human monocytes.

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.

Induction of tumor necrosis factor synthesis in human monocytes treated by transcriptional inhibitors.

Human blood monocytes and lymphocytes were separated by Percoll gradient fractionation. The synthesis of RNA was inhibited by actinomycin D (AcD) or alpha-amanitin (Amn). Monocytes were stimulated with LPS, lymphocytes were stimulated with phytohaemagglutinin (PHA). The activity of tumor necrosis factor (TNF-alpha) and lymphotoxin (LT) was measured by L-929 cell assay. It was shown that induction of TNF-alpha synthesis by LPS was not blocked by AcD and Amn. In contrast, the production of LT was blocked in cultures of lymphocytes treated by the inhibitors. Moreover, TNF-alpha synthesis was induced in resting monocyte cultures by AcD. Cycloheximide (Cy) inhibited the production of TNF-alpha. The data imply that TNF-alpha synthesis by human blood monocytes can be induced by posttranscriptional regulation of TNF-alpha mRNA presynthesized in vivo.