Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes.

Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1beta], IL-6, tumor necrosis factor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF-alpha-producing monocytes after 8 h of culture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1beta, IL-6, and TNF-alpha was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin.     

Intrinsic capacity of monocytes to produce cytokines ex vivo in patients with acute lymphoblastic leukaemia

Monocytic cytokine profiles of fifteen children with acute lymphoblastic leukaemia (ALL) were included to determine whether malignancy per se contributes to impaired cytokine profiles in vivo and ex vivo. The ex vivo tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) production was positively correlated with the monocyte number and with the number of intracellular TNF-alpha or IL-1beta positive cells in lipopolysaccharide (LPS)-stimulated MNC cultures. The mean ex vivo TNF-alpha and IL-1beta production per 1x10(4)monocytes in these cultures was not significantly different in children at diagnosis of ALL, at remission or in controls. High IL-10 plasma levels at diagnosis of ALL had no effect on the ex vivo TNF-alpha and IL-1beta production of monocytes in LPS stimulated MNC cultures. These results show that monocytes of ALL patients have a normal intrinsic capacity to produce cytokines ex vivo. However, the decreased monocyte number is responsible for the lower TNF-alpha and IL-1beta concentrations ex vivo upon LPS stimulation.     

Suppressive activity of a macrolide antibiotic, roxithromycin on co-stimulatory molecule expression on mouse splenocytes in vivo

The influence of roxithromycin (RXM) on the expression of co-stimulatory molecules, CD40, CD80 and CD86, was examined in vivo. When BALB/c mice were immunized intraperitoneally with two doses of dinitrophenylated ovalbumin (DNP-OVA) at 1 week intervals, intraperitoneal administration of RXM at 250 microg/kg once a day for 14 days strongly suppressed IgE contents in sera obtained from mice 22 days after the first immunization. In addition, RXM treatment of mice suppressed endogenous IL-4 contents in aqueous spleen extracts, which were enhanced by DNP-OVA immunization. We next examined the influence of RXM on co-stimulatory molecule expression on splenic lymphocytes. RXM treatment of the immunized mice caused suppression of CD40 expression, but this treatment did not affect CD80 and CD86 expression.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Production of pro- and anti-inflammatory cytokines by monocytes from patients with paracoccidioidomycosis

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.     

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.     

Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages

The eotaxins are a family of CC chemokines that coordinate the recruitment of inflammatory cells, in particular eosinophils, to sites of allergic inflammation. The cDNA for eotaxin-2 (CC chemokine ligand 24) was originally isolated from an activated monocyte library. In this study, we show for the first time that peripheral blood monocytes generate bioactive eotaxin-2 protein constitutively. Eotaxin-2 production was significantly up-regulated when monocytes were stimulated with the proinflammatory cytokine IL-1beta and the microbial stimuli, LPS and zymosan. In contrast, the Th2 cytokines, IL-4 and IL-13, and the proinflammatory cytokine, TNF-alpha, acting alone or in combination, did not enhance the generation of eotaxin-2 by monocytes. Indeed, IL-4 suppressed the generation of eotaxin-2 by LPS-stimulated monocytes. Although other chemokines, including macrophage-inflammatory protein-1alpha, monocyte chemoattractant protein-1, macrophage-derived chemokine, and IL-8 were generated by monocytes, eotaxin-1 (CC chemokine ligand 11) could not be detected in the supernatants of monocytes cultured in the presence or absence of any of the stimuli used in the above experiments. Furthermore, human dermal fibroblasts that produce eotaxin-1 did not generate eotaxin-2 under basal conditions or when stimulated with specific factors, including IL-4, IL-13, TNF-alpha, and LPS. When monocytes were differentiated into macrophages, their constitutive generation of eotaxin-2 was suppressed. Moreover, IL-4, but not LPS, up-regulated the production of eotaxin-2 by macrophages. Taken as a whole, these results support a role for macrophage-derived eotaxin-2 in adaptive immunity, with a Th2 bias. In contrast, a role for monocyte-derived eotaxin-2 is implicated in innate immunity.     

Effect of catecholamines on intracellular cytokine synthesis in human monocytes.

Proinflammatory cytokines produced by monocytes, like Interleukin-6 (IL-6), Interleukin-8 (IL-8), and tumor necrosis factor (TNF-alpha) are known for their pivotal role in the initiation of the inflammatory response following cardiopulmonary bypass (CPB). Catecholamines like epinephrine (Epi) and norepinephrine (Nor) are often necessary to stabilize the cardiac function in the early postoperative period and may influence the cytokine expression in monocytes. In this study we investigated the effects of Epi and Nor on IL-6, IL-8 and TNF-alpha expression in human monocytes stimulated with lipopolysaccharide (LPS) in whole blood, analyzed intracellularly by flow cytometry. Kinetics of intracellular proinflammatory cytokine production and LPS ED(50) were obtained. To simulate different stages of inflammation in vivo, varying concentrations of LPS (0.2 ng/ml, 1 ng/ml and 10 ng/ml) were used for stimulation. After a stimulation with LPS TNF-alpha was the first produced cytokine, followed by IL-8 and IL-6. All cytokines peaked from 3 h to 6 h. Epi and Nor had comparable effects on the expression of IL-6, IL-8 and TNF-a in monocytes. Both inhibited IL-6 and TNF-alpha expression in a concentration dependent manner whereas IL-8 expression remained unchanged. We conclude that monocytes are targets for Epi and Nor concerning their cytokine expression. The inhibiting effects of Nor and Epi were almost identical for all cytokines. Cytokine expression was affected most at low LPS concentrations.     

TNF-alpha and IL-1 beta are not essential to the inflammatory response in LPS-induced airway disease

To determine the role of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta in the lower respiratory tract inflammatory response after inhalation of lipopolysaccharide (LPS), we conducted inhalation exposure studies in mice lacking expression of TNF-alpha and/or IL-1 receptor type 1 and in mice with functional blockade of these cytokines using adenoviral vector delivery of soluble receptors to one or both cytokines. Alterations in airway physiology were assessed by pulmonary function testing before and immediately after 4 h of LPS exposure, and the cellular inflammatory response was measured by whole lung lavage and assessment of inflammatory cytokine protein and mRNA expression. Airway resistance after LPS exposure was similarly increased in all groups of mice without evidence that blockade of either or both cytokines was protective from this response. Additionally, all groups of mice demonstrated significant increases in lung lavage fluid cellularity with a complete shift in the population of cells to a predominantly neutrophilic infiltrate as well as elevation in inflammatory cytokine protein and mRNA levels. There were no significant differences between the groups in measures of lung inflammation. These results indicate that TNF-alpha and IL-1 beta do not appear to have an essential role in mediating the physiological or inflammatory response to inhaled LPS.     

Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ≤4 h, with TNF-alpha suppression preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta was non-detectable in cultures treated with SPIR prior to LPS, whereas elevated IL-1beta levels were seen when SPIR was added after LPS-stimulation. It is possible that the extracellular accumulation of IL-1beta is due to an increased release of already produced IL-1beta as a result of cell death. In conclusion, suppression of cytokine production by SPIR may be associated with its apoptotic potential, either directly (apoptosis is a consequence of suppressed cytokine production, or vice-versa) or indirectly (suppressed cytokine production and apoptosis are parallel but otherwise unrelated phenomena.)     

Lipopolysaccharide induces rapid production of IL-10 by monocytes in the presence of apoptotic neutrophils

There is growing evidence that apoptotic neutrophils have an active role to play in the regulation and resolution of inflammation following phagocytosis by macrophages and dendritic cells. However, their influence on activated blood monocytes, freshly recruited to sites of inflammation, has not been defined. In this work, we examined the effect of apoptotic neutrophils on cytokine production by LPS-activated monocytes. Monocytes stimulated with LPS in the presence of apoptotic neutrophils for 18 h elicited an immunosuppressive cytokine response, with enhanced IL-10 and TGF-beta production and only minimal TNF-alpha and IL-1beta cytokine production. Time-kinetic studies demonstrated that IL-10 production was markedly accelerated in the presence of apoptotic neutrophils, whereas there was a sustained reduction in the production of TNF-alpha and IL-1beta. This suppression of proinflammatory production was not reversible by depletion of IL-10 or TGF-beta or by addition of exogenous IFN-gamma. It was demonstrated, using Transwell experiments, that monocyte-apoptotic cell contact was required for induction of the immunosuppressive monocyte response. The response of monocytes contrasted with that of human monocyte-derived macrophages in which there was a reduction in IL-10 production. We conclude from these data that interaction between activated monocytes and apoptotic neutrophils creates a unique response, which changes an activated monocyte from being a promoter of the inflammatory cascade into a cell primed to deactivate itself and other cells.     

Lipopolysaccharide-induced cytokine cascade and lethality in LT alpha/TNF alpha-deficient mice.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is often considered the main proinflammatory cytokine induced by lipopolysaccharide (LPS) and consequently the critical mediator of the lethality associated with septic shock. MATERIALS AND METHODS: We used mice carrying a deletion of both the lymphotoxin alpha (LT-alpha) and TNF-alpha genes to assess the role of TNF in the cytokine cascade and lethality induced by LPS. RESULTS: Initial production of IL-1 alpha, IL-1 beta, IL-6, and IL-10 is comparable in wild-type and mutant mice. However, at later times, expression of IL-1 alpha, IL-1 beta, and IL-10 is prolonged, whereas that of IL-6 decreases in mutant mice. Expression of IFN-gamma is almost completely abrogated in mutants, which is in agreement with a more significant alteration of the late phase of the cytokine cascade. We measured similar LD50 (600 micrograms) for the intravenous injection of LPS in mice of the three genotypes (+/+, +/-, -/-), demonstrating that the absence of TNF does not confer long-term protection from lethality. However, death occurred much more slowly in mutant mice, who were protected more efficiently from death by CNI 1493, an inhibitor of proinflammatory cytokine production, than were wild-type mice. DISCUSSION: Thus, while TNF-alpha is not required for the induction of these cytokines by LPS, it modulates the kinetics of their expression. The lethality studies simultaneously confirm a role for TNF as a mediator of early lethality and establish that, in the absence of these cytokines, other mediators take over, resulting in the absence of long-term protection from LPS toxicity.     

Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

Participation of beta-adrenergic receptors on macrophages in modulation of LPS-induced cytokine release

For several years it is known that beta-adrenergic receptor agonists have anti-inflammatory effects. However, little is known about the role of beta-adrenergic receptors on macrophages in the modulation of cytokine production by beta-agonists during inflammation. In this study, the presence of beta-receptors on PMA-differentiated U937 human macrophages, and the participation of these receptors in the modulation of LPS-mediated cytokine production by beta-agonists was investigated. Total beta-receptor expression on undifferentiated (monocyte) and PMA-differentiated U937 cells was established using receptor binding studies on membrane fractions with a radio ligand. The expression of beta-receptors proved to be significantly lower on monocytes than on macrophages, additionally a predominant expression of beta 2-receptors was found. Production of the cytokines TNF-alpha, IL-6, and IL-10 by LPS-stimulated differentiated U937 cells was measured in time. Peak concentrations for TNF-alpha, IL-6 and IL-10 occurred at 3, 12 and 9 hrs, respectively. When differentiated U937 cells were incubated with both LPS and the beta-agonist clenbuterol the production of TNF-alpha and IL-6 was significantly reduced. However the production of IL-10 was increased. To study the mechanism of modulation of cytokine production in more detail, U937 macrophages were incubated with LPS/clenbuterol in combination with selective beta 1- and beta 2-antagonists. These results indicated that the beta 2- and not the beta 1-receptor is involved in the anti-inflammatory activity of clenbuterol.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

Differential regulation of TNF-alpha and IL-1beta production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors

The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-alpha and IL-1beta production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-alpha production, but only partially inhibited IL-1beta at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-alpha, but had no effect on IL-1beta production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-alpha and IL-1beta production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.     

Elastin receptor (spliced galactosidase) occupancy by elastin peptides counteracts proinflammatory cytokine expression in lipopolysaccharide-stimulated human monocytes through NF-kappaB down-regulation

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-gamma) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-alpha, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-kappaB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-kappaB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.