Repeated lipopolysaccharide (LPS) exposure inhibits HIV replication in primary human macrophages

Repetitive exposure of macrophages to microbial antigen is known to tolerize them to further stimulation and to inhibit proinflammatory cytokine release. Using transgenic (Tg) mice that incorporate the entire HIV-1 genome we have previously shown that toll like receptor (TLR)-2, -4, and -9 ligands induced tolerance as assessed by decreased proinflammatory cytokine secretion and nuclear factor-kappa beta activation. Yet, despite cytokine modulation, HIV-1 p24 production was enhanced in tolerized cells in vitro and in vivo. Since mice are not natural hosts for HIV infection, in the following report we examined whether TLR2 and TLR4 ligands induced tolerance in human monocytic cell lines stably expressing the HIV-long terminal repeat (LTR) luciferase construct (THP-LTR-Luc) as well as in primary macrophages that had been infected with HIV(BAL)in vitro. In THP-LTR-luc, TLR2 and TLR4 tolerization suppressed tumor necrosis factor (TNF)-alpha release and HIV-LTR transactivation. In HIV(BAL) infected macrophages, repeated LPS exposure inhibited HIV replication as assessed by decreased genetic expression and protein production of HIV-1 p24, although TNF-alpha release was not inhibited. These observations may have important clinical implications in understanding the role of macrophages as HIV reservoirs at anatomical sites where there is repeated exposure to microbial antigens.     

Arachidonic acid metabolism in alveolar macrophages. A comparison of cells from healthy subjects, allergic asthmatics, and chronic bronchitis patients

Arachidonic acid (AA) metabolism was studied in preparations of purified human alveolar macrophages (AM) from healthy subjects (HS = 5), allergic asthmatics (ABA = 9) and chronic bronchitis patients (CB = 7). AM incubated for 6 to 24 h in the presence of labeled AA and for an additional 5 h without labeled AA, released cyclooxygenase and lipoxygenase products into the medium. The study of the metabolites showed that the most abundant sulfidopeptide-leukotriene, was LTD4 as analyzed by TLC and identified by reversed phase HPLC. The release of LTD4 was time-dependent but it was shown to be significantly higher (p less than 0.01) in AM from ABA or CB than in those from HS. TLC analysis of radioactivity distributed between the different lipid classes at 24 h revealed more labeling in AM phospholipids from ABA and CB than in those from HS, and was reflected in phosphatidylethanolamine and phosphatidylinositol species. After 5 h without labeled AA the distribution was marked, by different in triglycerides, with a greater proportion of radioactivity in the control cells than in the pathological macrophages. Thus, the pathological lung state is an important factor affecting the release of LTD4 and the distribution of AA into cellular phospholipids. The differences observed between HS and ABA or CB phospholipid distribution suggests the existence of 2 different sources of AA release, one for inflammatory macrophages and another for quiescent cells.     

Role of transforming growth factor-beta(1) in down-regulating TNF production by alveolar macrophages during asbestos-induced pulmonary fibrosis

Activation of alveolar macrophages (AM) for tumour necrosis factor production is suppressed initially during the inflammatory response to fibrogenic dusts. We investigated the mechanisms involved in TNF suppression, notably the role of other AM-derived mediators including prostaglandin E(2) (PGE(2)), transforming growth factor-beta(1) (TGF-beta(1)), and interleukin 6 (IL-6). The action of PGE(2) and TGF-beta(1), on AM was different. At physiologically relevant doses (25-300 pg/ml), PGE(2) did not cause significant inhibition of Hpopolysaccharide (Lps)-induced TNF release by AM in vitro but stimulated IL-6 (up to six fold), an inhibitor of AM-derived TNT. In contrast, TGF-beta(1) (0.5-50 ng/ml) inhibited both LPS-induced TNT and IL-6 release by 50% but had no effect on PGE(2) production by AM. To determine the respective contribution of these different inhibitors in TNF suppression, AM from rats exposed to fibrogenic asbestos for weeks were treated with neutralizing antibody against TGF-beta(1) or indomethacin, an inhibitor of PGE(2) synthesis. Treatment of rat AM with anti-TGF-beta(1) but not indomethacin, abrogated the observed TNT suppression. These results suggest that an autocrine, TGF-beta(1)-dependent mechanism is involved in the down-regulation of TNF production by rat AM from animals with lung fibrosis.     

Role of IgE immune complexes in the regulation of HIV-1 replication and increased cell death of infected U1 monocytes: involvement of CD23/Fc epsilon RII-mediated nitric oxide and cyclic AMP pathways.

BACKGROUND: IgE/anti-IgE immune complexes (IgE-IC) induce the release of multiple mediators from monocytes/macrophages and the monocytic cell line U937 following the ligation of the low-affinity Fc epsilon receptors (Fc epsilon RII/CD23). These effects are mediated through an accumulation of cAMP and the generation of L-arginine-dependent nitric oxide (NO). Since high IgE levels predict more rapid progression to acquired immunodeficiency syndrome, we attempted to define the effects of IgE-IC on human immunodeficiency virus (HIV) production in monocytes. MATERIALS AND METHODS: Two variants of HIV-1 chronically infected monocytic U1 cells were stimulated with IgE-IC and virus replication was quantified. NO and cAMP involvement was tested through the use of agonistic and antagonistic chemicals of these two pathways. RESULTS: IgE-IC induced p24 production by U1 cells with low-level constitutive expression of HIV-1 mRNAs and extracellular HIV capsid protein p24 levels (U1low), upon their pretreatment with interleukin 4 (IL-4) or IL-13. This effect was due to the crosslinking of CD23, as it was reversed by blocking the IgE binding site on CD23. The IgE-IC effect could also be mimicked by crosslinking of CD23 by a specific monoclonal antibody. p24 induction by IgE-IC was then shown to be due to CD23-mediated stimulation of cAMP, NO, and tumor necrosis factor alpha (TNF alpha) generation. In another variant of U1 cells with > 1 log higher constitutive production of p24 levels (U1high), IgE-IC addition dramatically decreased all cell functions tested and accelerated cell death. This phenomenon was reversed by blocking the nitric oxide generation. CONCLUSIONS: These data point out a regulatory role of IgE-IC on HIV-1 production in monocytic cells, through CD23-mediated stimulation of cAMP and NO pathways. IgE-IC can also stimulate increased cell death in high HIV producing cells through the NO pathway.     

Platelet-activating factor (PAF) enhances tumor necrosis factor production by alveolar macrophages. Prevention by PAF receptor antagonists and lipoxygenase inhibitors

The effect of platelet-activating factor (PAF) on TNF production by rat alveolar macrophages (AM) and the role of endogenous leukotriene B4 (LTB4) in this regulation were examined. When AM were cultured with PAF alone, no change in TNF production was observed. However, the concomitant addition of PAF and muramyl dipeptide to AM cultures markedly enhanced (2- to 3-fold) TNF production in a concentration-dependent fashion with peak effect at 10(-10)M PAF. This enhancement occurred when muramyl dipeptide and PAF were present together at the initiation of the 24-h culture. Stimulation of TNF production by PAF was blocked by specific, but structurally different PAF receptor antagonists, BN 52021, CV3988 and WEB 2086. Additionally, the stereoisomer of PAF, [S]PAF, and the biologically inactive precursor/metabolite of PAF, lyso-PAF failed to induce significant enhancement in TNF production. In parallel, addition of PAF to AM triggered LTB4 release in a concentration-dependent manner. Inhibition of 5-lipoxygenase by nordihydro-guaiaretic acid or AA-861 blocked the PAF-induced augmentation of both TNF and LTB4 production. This was partially reversed by addition of exogenous LTB4. Collectively, these data suggest that PAF enhances TNF production by interaction with a specific putative receptor and by subsequent induction of endogenous 5-lipoxygenase activity in AM.     

Intrapulmonary TNF gene therapy reverses sepsis-induced suppression of lung antibacterial host defense

Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia. Although TNF-alpha (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung. Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens. To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of PSEUDOMONAS: aeruginosa. In animals undergoing sham surgery followed by bacterial challenge, PSEUDOMONAS: were nearly completely cleared from the lungs by 24 h. In contrast, mice undergoing CLP were unable to clear P. aeruginosa and rapidly developed bacteremia. Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals. Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with PSEUDOMONAS: from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector. Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo. These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense.     

Alveolar macrophages differ from blood monocytes in human IL-1 beta release. Quantitation by enzyme-linked immunoassay

Fresh human alveolar macrophages and blood monocytes were stimulated with LPS and assessed for their ability to produce and release antigenic IL-1 beta. Using a sensitive and specific ELISA for IL-1 beta, monocytes released 13.3 +/- 3.1 ng/10(6) cells compared to 3.5 +/- 0.8 ng/10(6) cells for alveolar macrophages (p less than 0.01). To investigate the reason for this difference in IL-1 beta release, monocytes were compared to alveolar macrophages for total IL-1 beta production (i.e., the amount released plus that detected in the lysates). Monocytes produced a total of 19.0 +/- 3.2 ng/10(6) cells whereas alveolar macrophages produced 24.8 +/- 5.6 ng/10(6) cells (p = 0.37). The relative increase in alveolar macrophage intracellular IL-1 beta was confirmed by Western blot analysis of cell lysates. Thus, the limitation in IL-1 release from alveolar macrophages appears to be due to a decrease in the processing and release of the IL-1 beta precursor. In addition, TNF production studies demonstrated that the limitation in IL-1 release was not a generalized defect. In contrast to the IL-1 beta data, when TNF was measured from monocytes and macrophages, monocytes released only 14.6 +/- 3.4 ng/10(6), whereas macrophages released 101 +/- 30 ng/10(6) (p less than 0.02). In this same context, when fresh monocytes were allowed to mature in vitro they took on monokine production characteristics similar to alveolar macrophages. In vitro matured monocytes had a greater than 20-fold decrease in their ability to release IL-1 beta and a 6- to 8-fold increase in their ability to release TNF. Taken together, these studies suggest that IL-1 beta release is limited in mature mononuclear phagocytes as compared to fresh blood monocytes, and furthermore, that IL-1 beta regulation differs significantly from that of TNF-alpha.     

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.     

Alveolar macrophages from systemic sclerosis patients: evidence for IL-4-mediated phenotype changes

The mechanism of chronic lung inflammation leading to lung fibrosis is unknown and does not have a characteristic inflammatory macrophage phenotype. This study was undertaken to determine whether a change in macrophage phenotype could account for chronic lung inflammation. In this study, human alveolar macrophages (AM) from subjects with systemic sclerosis (SSc) were obtained from bronchoalveolar lavage (BAL) and characterized on the basis of function (response to LPS), phenotype, and relative cell-surface B7 expression. AM from the subjects' disease-involved and noninvolved lung lobes were compared with each other and to AM from normal volunteer BAL. AM from involved SSc lobes produced significantly more interleukin (IL)-1beta and PGE(2) than AM from uninvolved lobes in response to LPS, but there was no spontaneous production of either mediator. The activator AM phenotype designated by RFD1+ surface epitope was significantly elevated in SSc BAL samples compared with normal BAL, although there were no differences comparing involved vs. noninvolved lobes within SSc subjects. The major histocompatibility complex II costimulatory molecule B7.2 was also significantly elevated in SSc AM compared with normal AM, again with no differences between involved and noninvolved lobes. In an attempt to determine environmental influences on AM phenotypes, normal AM were cultured in vitro with IFN-gamma, IL-3, IL-4, IL-10, IL-12, or dexamethasone for 6 days. Of the cytokines examined, only IL-4 induced significant increases in both the activator phenotype RFD1+ and B7.2 expression. Taken together, these results indicate that IL-4 could account for proinflammatory AM phenotype changes and B7 surface-marker shifts, as seen in subjects with SSc.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.

BACKGROUND: We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment. MATERIALS AND METHODS: The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis. RESULTS: The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients. CONCLUSIONS: The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

Alpha interferon suppresses virion but not soluble human immunodeficiency virus antigen production in chronically infected T-lymphocytic cells.

Alpha interferon (IFN-alpha) is effective in preventing the release of human immunodeficiency virus (HIV) from chronically infected T-lymphocytic (ACH-2) and promonocytic (U1) cell lines stimulated with the phorbol ester phorbol-12-myristate-13 acetate (PMA). In the present study, we observed that together with particle production, shedding of HIV antigen (p24gag) occurs in the T-cell line ACH-2 both constitutively and after stimulation with PMA. IFN-alpha, although effective in suppressing the release of HIV particles, did not inhibit shedding of p24gag into the culture supernatants of either unstimulated or PMA-stimulated cells. These observations may be of relevance in the evaluation of the in vivo efficacy of IFN-alpha treatment of HIV-infected individuals as determined by levels of p24 antigen in plasma.     

Pro-inflammatory and pro-oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17 beta-estradiol

Microglia are activated in humans following infection with human immunodeficiency virus (HIV), and brain inflammation is thought to be involved in neuronal injury and dysfunction during HIV infection. Numerous studies indicate a role for the HIV regulatory protein Tat in HIV-related inflammatory and neurodegenerative processes, although the specific effects of Tat on microglial activation, and the signal transduction mechanisms thereof, have not been elucidated. In the present study, we document the effects of Tat on microglial activation and characterize the signal transduction pathways responsible for Tat's pro-inflammatory effects. Application of Tat to N9 microglial cells increased multiple parameters of microglial activation, including superoxide production, phagocytosis, nitric oxide release and TNF alpha release. Tat also caused activation of both p42/p44 mitogen activated protein kinase (MAPK) and NF kappa B pathways. Inhibitor studies revealed that Tat-induced NF kappa B activation was responsible for increased nitrite release, while MAPK activation mediated superoxide release, TNF alpha release, and phagocytosis. Lastly, pre-treatment of microglial cells with physiological concentrations of 17 beta-estradiol suppressed Tat-mediated microglial activation by interfering with Tat-induced MAPK activation. Together, these data elucidate specific components of the microglial response to Tat and suggest that Tat could contribute to the neuropathology associated with HIV infection through microglial promulgation of oxidative stress.     

Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

Background

Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM₁₀) in vitro increase their production of inflammatory mediators and that supernatants from PM₁₀-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.

Methods

The present study concerns co-culture of AM and HBEC exposed to PM₁₀ (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.

Results

AM/HBEC co-cultures exposed to 100 μg/ml of PM₁₀ for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM₁₀-induced increase in co-culture mRNA expression.

Conclusion

We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM₁₀-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.     

Adrenomedullin release in the rat mesenteric resistance artery

Adrenomedullin (AM) is a potent vasodilator peptide whose major source is the vascular wall. In the present study, the mechanism of release of AM was investigated in the rat mesenteric resistance artery. The isolated mesenteric vascular bed was perfused with Krebs solution at a constant flow rate (5 ml/min) and AM in the perfusate was measured by a highly sensitive enzyme immunoassay (Immunoenzymometric assay; IEMA) method. In preparations without endothelium, spontaneous release of AM was detected in the perfusate (68.7+/-5.8 fmol/ml, n=45). Periarterial nerve stimulation (PNS, 4 and 8 Hz) caused 11.4+/-3.9% (4 Hz) and 9.1+/-3.5% (8 Hz) decreases in the spontaneous release of AM. Removal of Ca2+ from the medium did not affect the spontaneous AM release, but abolished the PNS-induced inhibition of spontaneous AM release. Perfusion of 10nM calcitonin gene-related peptide (CGRP) or 0.1 microM capsaicin (inducer of CGRP release) inhibited significantly the spontaneous AM release. PNS (8 Hz)-induced inhibition of spontaneous AM release was antagonized by CGRP(8-37) (CGRP receptor antagonist). These results suggest that AM is mainly released from vascular smooth muscle cells of the rat mesenteric artery and endogenous or exogenous CGRP inhibits AM release.     

Reduced glucocorticoid sensitivity of monocyte interleukin-6 release in male employees with high plasma levels of tumor necrosis factor-alpha

Cytokine production by monocytes plays a key role in atherosclerosis. In vitro, preincubation of whole blood with tumor necrosis factor (TNF)-alpha regulates interleukin (IL)-6 release from monocytes stimulated with lipopolysaccharide (LPS). We investigated whether plasma levels of TNF-alpha would also relate to LPS-stimulated monocyte IL-6 production and the inhibitory effect of a glucocorticoid on this process. 224 middle-aged men were assigned to three groups according to tertiles of plasma levels of TNF-alpha. Subjects in the highest tertile (high TNF-alpha, n = 75) were compared to those in the lowest (low TNF-alpha, n = 74) and medium tertile (medium TNF-alpha, n = 75), respectively. In vitro monocyte IL-6 release following lipopolysaccharide (LPS)-stimulation was assessed with and without coincubation with incremental doses of dexamethasone. Monocyte glucocorticoid sensitivity was defined as the dexamethasone concentration inhibiting IL-6 release by 50%. Subjects with high TNF-alpha showed more IL-6 release after LPS-stimulation than those with low TNF-alpha (p =.011). Monocyte glucocorticoid sensitivity was lower in subjects with high levels of TNF-alpha than in subjects with low (p =.014) and with medium (p =.044) levels of TNF-alpha. Results held significance when a set of classic cardiovascular risk factors was controlled for. Our findings suggest that elevated plasma levels of TNF-alpha might enhance LPS-stimulated IL-6 release from circulating monocytes. Such a mechanism might contribute to exaggerated monocyte cytokine release in vivo to any LPS-like danger signal such as related to an infection or cellular stress thereby promoting atherosclerosis.     

Neurotensin enhances IL-1 production by activated alveolar macrophages

Peptides may play a physiologic role in regulating immune responses and in triggering a variety of cellular events that modify the sensitivity of cells in the periphery. Neurotensin (NT) is present in the lung and it has been shown to bind to mouse peritoneal macrophages and influence their phagocytic ability. In this study, the effect of NT on the production of IL-1 by rat alveolar macrophages (AM) has been investigated. Although NT did not stimulate the release of IL-1 or increase the apparent intracellular pool of IL-1 when incubated with AM, there was significant cell changes, such as increased adherence, spreading, and altered shape. Furthermore, when AM were stimulated with LPS, both the intracellular and extracellular pools of IL-1 were significantly increased by NT. This effect was dose dependent and was observed at concentrations ranging from 10(-11) to 10(-6) M. NT did not modify the kinetics of LPS-induced IL-1 release nor the effects of a given suboptimal concentration of LPS. The release of IL-1 by various inducers, including muramyl dipeptide (MDP) and zymosan was also enhanced by NT, suggesting a general modulator role for this neuropeptide. When NT was added concomitantly with other potentiators of IL-1 production, such as IFN-gamma and leukotriene B4, no synergistic effect on IL-1 release was seen. Kinetics experiments showed that optimal enhancement of IL-1 production occurred when AM cultures were preincubated with NT before addition of MDP or when NT and MDP were present together at the initiation of the 24-h AM cultures. Taken together, our data suggest that NT acts early in the induction process of IL-1. Because IL-1 plays an important role both in the initiation of the immune response and in the local manifestations of inflammation, NT released in the vicinity of pulmonary blood vessels and the respiratory epithelium may modulate immunologically relevant responses in the lung microenvironment.     

Alveolar macrophages from HIV-infected patients with pulmonary tuberculosis retain the capacity to respond to stimulation by lipopolysaccharide

The functional capacity of alveolar macrophages (AM) in human immunodeficiency virus (HIV)-infected patients with pulmonary tuberculosis (TB) is not completely understood. To investigate the capacity of AM to mediate inflammatory responses, we obtained AM from human subjects by bronchoalveolar lavage (BAL) and studied the cells ex vivo. We compared AM from HIV-infected patients with suspected pulmonary TB to AM from healthy, HIV-negative controls for their capacity to produce TNF-alpha or IL-6 spontaneously and upon stimulation with lipopolysaccharide (LPS). Cytokine-producing cells were identified by macrophage markers and intracellular cytokine staining and flow cytometry. A higher proportion of AM from patients with microbiologically confirmed pulmonary TB than patients with probable TB or controls spontaneously expressed TNF-alpha shortly after isolation (geometric means: 38.5%, 23.7% and 15.8%, respectively), suggesting endogenous cytokine production. The proportions of AM spontaneously expressing TNF-alpha positively correlated with peripheral blood CD4(+) T-lymphocyte counts in patients (partial r=0.60, p=0.003) but not controls. Stimulation with LPS resulted in a significant increase in the proportions of TNF-alpha- and IL-6-positive AM from patients and controls (p<0.01). Bronchoalveolar lavage fluid (BALF) from confirmed TB patients also contained higher concentrations of the inflammatory cytokines predominantly produced by macrophages, IL-6 and IL-8, than controls (geometric mean cytokine concentrations per gram of BALF albumin were 1291 pg/g vs. 115 pg/g, p=0.03 for IL-6 and 4739 pg/g vs. 704 pg/g, p=0.03 for IL-8). We concluded that AM from HIV-infected patients with pulmonary TB produced and released inflammatory cytokines in vivo and retained their innate ability to respond to stimulation by LPS.