Decreases in plasma TNF-alpha level and IFN-gamma mRNA level in peripheral blood mononuclear cells (PBMC) and an increase in IL-2 mRNA level in PBMC are associated with effective highly active antiretroviral therapy in HIV-infected patients

In this study, we investigated the cytokine profiles of 14 treatment-naive HIV-infected patients on the initiation of highly active antiretroviral therapy (HAART). At baseline, plasma levels of TNF-alpha and its mRNA in peripheral blood mononuclear cells (PBMC) were highest in the most severely immunocompromised patients (<200 CD4+ cells/mm3). After 12 months of HAART, the virus was undetectable in the plasma of all patients (<200 copies/ml), and median CD4 T cell counts had increased (+164 cells/mm3). We also observed a gradual decrease in the number of proviral DNA copies in PBMC and in immune activation, with lower levels of IFN-gamma mRNA in PBMC associated with weaker activation of CD8+ T cells and lower levels of plasma TNF-alpha. IL-2 mRNA levels in PBMC were found to increase in parallel. The decrease in TNF-alpha and IFN-gamma levels and the increase in IL-2 production appear to be correlated with the efficacy of HAART in naive immunocompromised HIV-infected individuals.     

Inflammatory responses in blood samples of human immunodeficiency virus-infected patients with pulmonary infections

We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1beta (IL-1beta), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-alpha) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-alpha, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1beta, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-alpha levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5).     

Lipopolysaccharide from an Escherichia coli htrB msbB mutant induces high levels of MIP-1 alpha and MIP-1 beta secretion without inducing TNF-alpha and IL-1 beta

OBJECTIVE: To identify a lipopolysaccharide (LPS) that retains the capacity to induce beta-chemokine secretion without the concomitant activation of pyrogenic cytokines. METHODS: LPS was extracted from strain MLK986 (mLPS), an htrB1::Tn10, msbB::ocam mutant of Escherichia coli that is defective for lipid A synthesis, and from wild-type parent E coli strains, W3110 (wtLPS). The capacity of these LPS preparations to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and macrophage inflammatory proteins 1 alpha (MIP-1 alpha) and MIP-1 beta was assessed using a human peripheral blood mononuclear cell (PBMC) activation assay. RESULTS: Stimulation of PBMCs with mLPS did not induce measurable levels of pyrogenic cytokines TNF-alpha and IL-1 beta, whereas wtLPS induced high levels of these cytokines. Furthermore, mLPS antagonized the induction of TNF-alpha secretion by wtLPS. Nonetheless, mLPS retained a discrete agonist activity that induced MIP-1 alpha and MIP-1 beta secretion by PBMCs. This latter agonist activity appears to be unique to mLPS, since two previously documented LPS antagonists, Rhodobacter sphaeroides diphosphoryl lipid A and synthetic lipid IVA, did not induce MIP-1 alpha and MIP-1 beta secretion. Furthermore, synthetic lipid IVA was an antagonist of MIP-1 alpha and MIP-1 beta induction by mLPS. CONCLUSION: These results show that mLPS exhibits a novel bipartite activity, being an effective antagonist of TNF-alpha induction by wtLPS, while paradoxically being an agonist of MIP-1 alpha and MIP-1 beta secretion.     

Neutrophils regulate the expression of cytokines, chemokines and nitric oxide synthase/nitric oxide in mice injected with Bothrops atrox venom

Bothrops atrox crude venom injected intraperitoneal (i.p.) into BALB/c mice induced local afflux of inflammatory cells, one neutrophil-rich peak after 6h and another macrophage-rich peak after 48 h. A similar pattern of local cell afflux plus edema, Delta lesions of some skeletal muscle cells, and hemorrhage were observed in mice intramuscular (i.m.) injected with the venom. Measurement of serum cytokines in neutrophil-depleted (by anti-mouse rat monoclonal antibody (mAb) RB6-8C5) and non-depleted BALB/c mice was performed by ELISA. With the exception of IL-1beta (78 pg/ml), higher levels of IL-6 (1348 pg/ml), MIP-1beta (437 pg/ml) and MIP-2 (904 pg/ml) were observed in neutrophil-depleted mice, in comparison to the values found in non-neutrophil depleted mice: IL-1beta (437 pg/ml), IL-6 (750 pg/ml), MIP-1beta (165 pg/ml) and MIP-2 (90 pg/ml). TNF-alpha was not detected. NO was detected (18 microM) 24h after venom injection in neutrophil-depleted mice. RT-PCR using representative primers detected expression of mRNA in cells from BALB/c mice injected with B. atrox venom: (a) for IL-1beta, IL-6, inducible nitric oxide synthase (iNOS), CXCR2, MIP-2 and RANTES in cells from mice that were neutrophil-depleted or not; (b) for CCR1, CCR5 and MIP-1beta in cells from neutrophil-depleted mice; (c) for MIP-1alpha in cells from non-neutrophil-depleted mice; (d) TNF-alpha and TGF-beta were not detected in either of the mice. These results indicate that neutrophils play a role in regulating the production of some cytokines and chemokines as well as locally expressed or liberated iNOS/NO in tissues injected with B. atrox crude venom.     

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.     

Pulmonary inflammation induced by Pseudomonas aeruginosa lipopolysaccharide, phospholipase C, and exotoxin A: role of interferon regulatory factor 1

Chronic pulmonary infection with Pseudomonas aeruginosa is common in cystic fibrosis (CF) patients. P. aeruginosa lipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta-6, gamma interferon (IFN-gamma), MIP-1 alpha MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-alpha and was a weak inducer of IL-1 beta, IL-6, macrophage inflammatory protein 1 alpha (MIP-1 alpha), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-gamma. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-alpha, IL-1 beta, and IFN-gamma than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1 beta and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1 alpha and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8(+) and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors from P. aeruginosa induce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.     

Concentrations and significance of cytokines and other immunologic factors in semen of healthy fertile men

BACKGROUND: The purpose of this study was to establish normal reference values for several immunologic factors in semen to provide a foundation for understanding their physiologic significance in health and disease. METHODS: Semen from 83 healthy, fertile men was assessed by Bio-Plex or enzyme-linked immunosorbent assay to determine quantities of immunoglobulin (Ig) isotypes, chemokines, cytokines and growth factors. We also enumerated polymorphonuclear granulocytes (PMN) by peroxidase staining to examine the association of inflammation with levels of these factors. RESULTS: High concentrations of IgG and IgA were detected in all samples. IgG concentrations were significantly higher than IgA concentrations (P < 0.0001). Likewise, two multifunctional growth factors, transforming growth factor-beta1 and interleukin (IL)-7, and three chemokines, stromal cell-derived factor-1alpha, monocyte chemotactic/chemoattractant protein-1 and IL-8, were present in high concentrations in all samples (medians >1000 pg/ml). Other soluble factors were detectable in low concentration (medians <150 pg/ml), either in a majority of samples [IL-1alpha and beta, IL-5, IL-6, IL-13, IL-17, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1beta, interferon (IFN)-alpha and granulocyte colony-stimulating factor (CSF)], or in a minority of samples (MIP-1alpha, IL-2, IL-10, IL-12, TNF-alpha, IFN-gamma and granulocyte-macrophage-CSF). PMN counts significantly correlated with IL-1beta, IL-6, TNF-alpha, MIP-1alpha, MIP-1beta, IL-13 and IgA concentrations. CONCLUSIONS: The semen of healthy, fertile men contains a broad array of immunologic factors. These normative values can serve as a foundation for future studies on the role of these factors in infertility, genital tract infections and other pathologic conditions.     

Effect of pentoxifylline on levels of pro-inflammatory cytokines during chronic hepatitis C

The cellular and humoral natural immune response induced by hepatitis C virus (HCV) is commonly unable to eradicate the virus. HCV is a highly mutable, hepatotropic RNA virus that causes acute and chronic hepatitis, an infection that involves the production of various cytokines. The aim of the study is to analyse the expression of pro-inflammatory cytokines IL-1beta, TNF-alpha, IFN-gamma and the chemokine CXCL8 (IL-8) in liver tissue and their expression and secretion in PBMC of patients with chronic hepatitis C (CHC), in response to pentoxyfilline (PTX). We studied six CHC patients, naive to treatment. Patients received PTX 400 mg twice a day/8 weeks. Pentoxyfilline resulted in decreased expression of mRNA of liver IL-1beta, TNF-alpha and IFN-gamma: 144.2 versus 83.5 molecules of IL-1beta (P < 0.05), TNF-alpha 194.3 versus 17.6 molecules (P = 0.03) and IFN-gamma 26.1 versus 0.5 molecules (P = 0.04). Following PTX, PBMC exhibited a decrease in IFN-gamma mRNA 12.2 versus 1.5 molecules (P = 0.028) and CXCL8 4.2 versus 2.5 molecules (P = 0.027). In PBMC, only the secretion of TNF-alpha was decreased 1109 versus 933.5 pg/ml, P = 0.046. Production of cytokines both locally (within the liver) and systemically (PBMC) may serve as biomarkers of the infection with hepatitis C. PTX inhibits the expression of several pro-inflammatory cytokines in the liver. These results indicate that it is worth exploring PTX in hepatitis in future clinical trials in nonresponders to antiviral treatment.     

Immunoglobulin D enhances the release of tumor necrosis factor-alpha, and interleukin-1 beta as well as interleukin-1 receptor antagonist from human mononuclear cells.

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper-IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and alpha 1-acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), and IL-1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL-1 beta, TNF-alpha, and IL-lra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose-dependent and showed a 30-fold (TNF-alpha) to almost 150-fold (IL-1 beta) increase at the highest concentration (50 mg/l), while AGP (750 micrograms/ml) only increased the cytokine secretion fourfold (TNF-alpha) to almost 30-fold (IL-1 beta). The effect of IgD on IL-1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5-fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL-1 beta and TNF-alpha, although the effect was most apparent for TNF-alpha. Apart from inducing IL-1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF-alpha, IL-1 beta and IL-1ra and thus may contribute to the pathogenesis of HIDS.     

Differential effect of serotonin on cytokine production in lipopolysaccharide-stimulated human peripheral blood mononuclear cells: involvement of 5-hydroxytryptamine2A receptors

In order to provide additional insight into the in vivo significance of serotonin [5-hydroxytryptamine (5-HT)] in inflammation, we examined its effect on the production of tumor necrosis factor (TNF)-alpha, IL-1alpha, IL-1beta, IL-6, IL-10 and IL-1 receptor antagonist in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). 5-HT inhibited TNF-alpha production and increased IL-1beta production in PBMC. The level of IL-1beta-converting enzyme/caspase-1 remained unchanged, suggesting that the effect of 5-HT is not directly related to the IL-1beta maturation process. TNF-alpha mRNA and IL-1beta mRNA content did not change in the presence of 5-HT. 5-HT did not have any effect on the production of other cytokines studied. The inhibitory effect of 5-HT on TNF-alpha production was antagonized by ketanserin, a selective 5-HT(2A) antagonist, and mimicked by DOI, a selective 5-HT(2A/2C) agonist. These findings suggest that the inhibition of TNF-alpha production by 5-HT involves the participation of the 5-HT(2A) receptor subtypes in PBMC. Accordingly, we detected the presence of 5-HT(2A) receptors in PBMC by Western blot analysis. Our data support a role of 5-HT in inflammation through its effect on cytokine production in PBMC.     

Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro

Ebola virus infection of humans is associated with high levels of circulating inflammatory chemokines and cytokines. We demonstrate that direct infection of human PBMC results in the induction of MCP-1, MIP-1alpha, RANTES, and TNF-alpha as early as 24 h p.i. in response to live virus. Monocyte-derived macrophages infected with live Ebola-virus secreted MIP-1alpha and TNF-alpha specifically while RANTES and MCP-1 were secreted by with both live or inactivated virus stimulation and do not require viral replication. Type I interferons (IFN-alpha and -beta), IL-1beta and IL-10, were not induced by Ebola virus. Furthermore, live virus infection of both PBMCs and monocytes-derived macrophages inhibited IFN-alpha induced by double-stranded RNA in vitro. These data provide the first direct evidence of a role for macrophages in the pathogenesis to Ebola virus and suggest that Ebola virus can inhibit cellular antiviral mechanisms mediated by type I interferons.     

Lactobacilli and streptococci induce inflammatory chemokine production in human macrophages that stimulates Th1 cell chemotaxis

Macrophages have a central role in innate-immune responses to bacteria. In the present work, we show that infection of human macrophages with Gram-positive pathogenic Streptococcus pyogenes or nonpathogenic Lactobacillus rhamnosus GG enhances mRNA expression of inflammatory chemokine ligands CCL2/monocyte chemoattractant protein-1 (MCP-1), CCL3/macrophage-inflammatory protein-1alpha (MIP-1alpha), CCL5/regulated on activation, normal T expressed and secreted, CCL7/MCP-3, CCL19/MIP-3beta, and CCL20/MIP-3alpha and CXC chemokine ligands CXCL8/interleukin (IL)-8, CXCL9/monokine induced by interferon-gamma (IFN-gamma), and CXCL10/IFN-inducible protein 10. Bacteria-induced CCL2, CCL7, CXCL9, and CXCL10 mRNA expression was partially dependent on ongoing protein synthesis. The expression of these chemokines and of CCL19 was dependent on bacteria-induced IFN-alpha/beta production. CCL19 and CCL20 mRNA expression was up-regulated by IL-1beta or tumor necrosis factor alpha (TNF-alpha), and in addition, IFN-alpha together with TNF-alpha further enhanced CCL19 gene expression. Synergy between IFN-alpha and TNF-alpha was also seen for CXCL9 and CXCL10 mRNA expression. Bacteria-stimulated macrophage supernatants induced the migration of T helper cell type 1 (Th1) cells, suggesting that in human macrophages, these bacteria can stimulate efficient inflammatory chemokine gene expression including those that recruit Th1 cells to the site of inflammation. Furthermore, L. rhamnosus-induced Th1 chemokine production could in part explain the proposed antiallergenic properties of this bacterium.     

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.     

CXCR4 and CCR5 expression by H9 T-cells is downregulated by a peptide-nucleic acid immunomodulator

Product R (Reticulose(TM)) is a peptide-nucleic acid immunomodulator with broad-spectrum antiviral activity that was recently shown to increase expression of mRNAs encoding the proinflammatory cytokines, IFN-gamma, IL-1beta, IL-6 and TNF-alpha. Since these cytokines induce expression of the chemokines, MIP-1alpha, MIP-1beta, RANTES, and SDF-1, all of which inhibit viral infectivity, we were interested to determine if Product R also alters chemokine expression. In addition, the finding, that Product R decreases HIV-1 RNA and extracellular p24 antigen in H9 T-lymphoma cells, suggested to us that this drug may block viral infection by reducing the expression of chemokine receptors on target cells. We have therefore utilized H9 cells to test the effects of Product R on expression of mRNAs encoding the chemokine receptors, CD4, CXCR4 and CCR5, as well as their ligands, IL-16, SDF-1, MIP-1alpha, MIP-1beta, and RANTES, by RT-PCR. We also assayed the effect of Product R on surface receptor expression by flow cytometry, and on the chemotactic activity of these cells towards the CXCR4 ligand, SDF-1, and the CCR5 ligands, MIP-1alpha and RANTES. H9 cells were cultured for 3-21 days in medium containing 5% or 10% Product R, or 5% or 10% PBS. We found that, compared to control cultures, cells cultured in media containing Product R expressed lower amounts of CXCR4 and CCR5 mRNA and surface antigen at all time points. Culture for 3 days in media containing Product R also reduced the ability of cells to migrate towards 10-20 ng/ml SDF-1 and 100-250 ng/ml RANTES. In contrast, Product R had no effect on the expression of CD4 mRNA and receptor protein, or on expression of IL-16 mRNA. These findings suggest that Product R may have clinical efficacy in HIV-1-infected patients by downregulating viral coreceptors on target T-cells.