TL1A/DR3 axis involvement in the inflammatory cytokine network during pulmonary sarcoidosis.

BACKGROUND: TNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place. METHODS: In this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects. RESULTS: Our results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1. CONCLUSIONS: These data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.

Rapid reconstitution of functionally active 6-sulfoLacNAc(+) dendritic cells (slanDCs) of donor origin following allogeneic haematopoietic stem cell transplant.

The role of dendritic cells (DCs) and macrophages in allogeneic haematopoietic stem cell transplant (HSCT) is critical in determining the extent of graft-versus-host response. The goal of this study was to analyse slanDCs, a subset of human proinflammatory DCs, in haematopoietic stem cell (HSC) sources, as well as to evaluate their 1-year kinetics of reconstitution, origin and functional capacities in peripheral blood (PB) and bone marrow (BM) of patients who have undergone HSCT, and their presence in graft-versus-host disease (GVHD) tissue specimens. slanDCs were also compared to myeloid (m)DCs, plasmacytoid (p)DCs and monocytes in HSC sources and in patients' PB and BM throughout reconstitution. slanDCs accounted for all HSC sources. In patients' PB and BM, slanDCs were identified from day +21, showing median frequencies comparable to healthy donors, donor origin and kinetics of recovery similar to mDCs, pDCs, and monocytes. Under cyclosporin treatment, slanDCs displayed a normal pattern of maturation, and maintained an efficient chemotactic activity and capacity of releasing tumour necrosis factor (TNF)-α upon lipopolysaccharide (LPS) stimulation. None the less, they were almost undetectable in GVHD tissue specimens, being present only in intestinal acute GVHD samples. slanDCs reconstitute early, being donor-derived and functionally competent. The absence of slanDCs from most of the GVHD-targeted tissue specimens seems to rule out the direct participation of these cells in the majority of the local reactions characterizing GVHD.

Interleukin 10 (IL-10) inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. Evidence for an autocrine role of tumor necrosis factor and IL-1 beta in mediating the production of IL-8 triggered by lipopolysaccharide.

In this study we have examined the effects of interleukin 10 (IL-10) on polymorphonuclear leukocytes (PMN), and found that it is a potent inhibitor of tumor necrosis factor (TNF), IL-1 beta, and IL-8 secretion triggered by lipopolysaccharide (LPS). Cytokine production by phagocytosing PMN was also inhibited by IL-10, but to a lesser extent than the LPS-induced production. As shown by Northern blot analysis, IL-10 diminished the levels of TNF, IL-1 beta, and IL-8 mRNAs late after the onset of stimulation of PMN with LPS. In addition, we provide evidence that the kinetics of LPS-induced IL-8 production by PMN is composed of two distinct phases. Specifically, our experiments demonstrated that in the first phase, the production of IL-8 is a process directly induced by LPS that lasts for some hours. After this early wave, a second phase begins that is sustained and leads to an elevated production of IL-8 that appears to be due to the endogenous release of TNF and IL-1 beta. This second wave can in fact be blocked by anti-TNF and anti-IL-1 beta neutralizing antibodies, and by IL-10 as the consequence of its downregulatory effects on TNF and IL-1 beta release. Taken together, these findings identify novel biological actions of IL-10 as a suppressor of the inflammatory response.

Interleukin 10 (IL-10) upregulates IL-1 receptor antagonist production from lipopolysaccharide-stimulated human polymorphonuclear leukocytes by delaying mRNA degradation.

Polymorphonuclear leukocytes (PMN) have been identified as cells capable of producing a number of pro- and antiinflammatory cytokines in response to specific agonists. Previously, we showed that tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and IL-8, are produced by PMN after stimulation with agonists, such as lipopolysaccharide (LPS). In this study, we demonstrate that LPS is also a potent stimulus for the mRNA expression and release of the IL-1 receptor antagonist (IL-1ra). In addition, we show that the release of IL-1ra from LPS-stimulated PMN is markedly potentiated in the presence of IL-10 (from two to threefold after 18 h of stimulation). Moreover, we observed that this upregulation of IL-1ra production by IL-10 in LPS-stimulated PMN took place through IL-1ra mRNA stabilization. Indeed, the half-life of IL-1ra mRNA was prolonged in PMN stimulated in the presence of IL-10 and LPS, as compared with cells stimulated with LPS alone. That IL-10 selectively upregulates IL-1ra production in LPS-activated PMN, while it inhibits the production of IL-1 beta, TNF, and IL-8 under the same conditions, suggests that IL-10 may be an important physiologic regulator of cytokine production from PMN, and emphasizes the potential role of IL-10 in inflammatory responses.

Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8.

After phagocytosis of yeast opsonized with IgG, neutrophil leukocytes (polymorphonuclear leukocytes [PMN]) expressed high levels of neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8) mRNA, which peaked after 3-5 h and were still elevated after 18 h. A similar but quantitatively less prominent effect was obtained with lipopolysaccharide (LPS). After phagocytosis, but not after exposure to LPS, the PMN progressively released considerable amounts of NAP-1/IL-8 into the culture medium (18.6-50 ng/ml in 18 h). The peptide released was biologically active, as indicated by the transient elevation of cytosolic-free calcium in PMN exposed to aliquots of the culture supernatants, and desensitization by prestimulation of the cells with recombinant NAP-1/IL-8. By producing NAP-1/IL-8 at sites where they phagocytose invading microorganisms, PMN could enhance the recruitment of new defense cells.

Fc gamma R(CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2+ in Fc gamma R(CD16)-induced transcription and expression of lymphokine genes.

In this study, we present evidence that interaction of Fc gamma R(CD16) with ligands (immune complexes or anti-CD16 antibodies) induces a rapid rise in [Ca2+]i and fast production of both inositol 1,4,5 triphosphate (IP3) and IP4 in homogeneous NK cell preparations. Part of the initial [Ca2+]i rise observed upon stimulation of NK cells with either anti-CD16 antibodies alone or after their crosslinking at the cell membrane depends on Ca2+ mobilization from intracellular stores, but sustained [Ca2+]i levels are maintained, after the initial spike, through influx of extracellular Ca2+. The [Ca2+]i rise is mediated, at least in part, by increases in IP3 after receptor-induced hydrolysis of membrane polyphosphoinositides (PPI). The role of extracellular Ca2+ in Fc gamma R(CD16)-dependent induction of lymphokine gene expression has been tested by evaluating production, mRNA accumulation and transcription of IFN-gamma and TNF in NK cells stimulated with Fc gamma R(CD16) ligands and/or rIL-2 in the presence of EGTA. Under these conditions, accumulation and transcription of both IFN-gamma and TNF mRNA induced by CD16 ligands, but not that induced by rIL-2, is completely abolished and neither cytokine can be detected at significant levels in the supernatant fluids of cells so treated. These data confirm that NK cell activation by specific ligands occurs through mechanisms distinct from those induced by IL-2, and indicate that extracellular Ca2+ represents a stringent requirement for cytokine production induced in NK cells through specific (Fc gamma R) stimulation. Our data also indicate that the [Ca2+]i rise induced upon Fc gamma R(CD16) crosslinking, though necessary, is not sufficient per se to induce activation of lymphokine genes, compatible with the hypothesis that Fc gamma R(CD16) crosslinking generates additional transducing signals that synergize with IL-2 to maximally activate NK cells.

Tumor necrosis factor and immune interferon synergistically induce cytochrome b-245 heavy-chain gene expression and nicotinamide-adenine dinucleotide phosphate hydrogenase oxidase in human leukemic myeloid cells.

Recombinant tumor necrosis factor (rTNF) and rIFN-gamma induce in the human leukemia cell lines HL-60, ML3, and U937 the accumulation of transcripts of the X chromosome-linked chronic granulomatous disease (X-CGD) gene, encoding the 91-kD heavy chain of cytochrome b-245, a component of the NADPH oxidase of phagocytic cells. The gene is induced within 6 h by either cytokine, and its accumulation is observed upon induction with rIFN-gamma up to 5 d. The combined effect of the two cytokines is more than additive. rIFN-gamma also induces accumulation of X-CGD mRNA in immature myeloid cells from peripheral blood of chronic myeloid leukemia (CML) patients, whereas rTNF has almost no effect. The cells from CML patients constitutively express TNF mRNA, suggesting that endogenously produced TNF may play a role in the effect of rIFN-gamma on these cells. rTNF induces X-CGD gene expression in the myeloid cell lines acting, at least in part, at the transcriptional level, as shown in nuclear run-on experiments. The gene encoding the 22-kD light chain of cytochrome b-245 is constitutively expressed in the human myeloid cell lines and the accumulation of its transcripts is affected by neither rTNF nor rIFN-gamma, rTNF and rIFN-gamma synergistically to induce the cell lines to express the cytochrome b-245 heterodimer (as evaluated by its visible spectrum), and to produce NADPH oxidase activity and H2O2 upon stimulation with phorbol diesters.

Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.

Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b.

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.

Different mechanisms of activation of proliferating CD3+ cells in patients with lymphoproliferative disease of granular lymphocytes.

To investigate the role of p55 and p75 chains of interleukin-2 receptor (IL-2R) on the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), the cells obtained from 11 LDGL patients (belonging to the CD3+ group) were studied for (a) the surface expression and (b) mRNA transcripts of the p55 and p75 IL-2R after activation with anti-CD3 monoclonal antibody (mAb) or interleukin-2 (IL-2). The effects of mAbs specifically blocking the p55 and p75 IL-2R on the generation of proliferative and cytotoxic functions were studied following anti-CD3 mAb stimulation. A significant difference was observed in the expression of p55 and p75 antigens on LDGL cells under resting conditions: a low number of p55 IL-2R+ (mean 1.2 +/- 0.4%) and high values of p75 IL-2R+ cells (54.9 +/- 7.4%). Accordingly, a barely detectable message for the p55 IL-2R and a strong signal for the p75 IL-2R mRNA were demonstrated. Following activation with anti-CD3 or IL-2, different patterns of IL-2R expression were observed. Anti-CD3 mAb induced an increase in the expression of the p55 IL-2R both at the mRNA and antigen level, whereas the p75 values remained consistently raised. In contrast, IL-2 induced the expression of p55 IL-2R mRNA associated with only a slight expression of this antigen. This finding was associated with a decrease in the cell expression of the p75 IL-2R, whereas the amount of p75 mRNA was unchanged. Both anti-CD3 mAb and IL-2 induced cell proliferation and cytotoxicity against the K-562 target cells. Anti-p55 IL-2R mAb did not affect the cytotoxic activity mediated by anti-CD3, but it markedly inhibited cell proliferation. Anti-p75 mAb did not inhibit either lytic function or cell proliferation mediated by anti-CD3 mAb, suggesting that only the high affinity IL-2R (p55 plus p75) is involved in anti-CD3 mediated cell activation in LDGL patients. This mechanism is different from that responsible for the IL-2 activation of CD3+ GL in LDGL patients, which is achieved through the p75 IL-2R alone. These results provide new insights into the pathophysiology of proliferating GL in LDGL patients and may also contribute to further characterization of the normal CD3+ GL population.

IL-8 mRNA expression and IL-8 production by acute myeloid leukemia cells.

Purified leukemic cells from 30 acute myeloid leukemia (AML) cases at diagnosis were investigated for the presence of interleukin 8 (IL-8) mRNA by Northern blot analysis. IL-8 specific transcripts were detected in uncultured blasts in 14/30 cases, 10/14 from patients with M4-M5 and 4/14 from cases with M0-M3 morphology. The transcript expression was associated with the detection of IL-8 molecule in blast cells by immunostaining performed on cytospin preparations. After 24-hour culture, a strong up-regulation or the appearance in cases negative before culture of IL-8 mRNA was observed in all cases tested, and culture supernatants contained high amounts of IL-8. Our data demonstrate that leukemic cells in AML are equipped with the functional apparatus for IL-8 production. Since IL-8 displays a wide range of biological activities, including the regulation of some membrane molecules relevant to adhesion and migration processes, its production by AML blasts might be of relevance for the pattern of leukemic growth.

Phagocytosis of opsonized yeast induces tumor necrosis factor-alpha mRNA accumulation and protein release by human polymorphonuclear leukocytes.

In this report we show that phagocytosis of yeast particles opsonized with IgG (Y-IgG) by human polymorphonuclear cells (PMN) results in the selective induction of tumor necrosis factor (TNF-alpha) messenger RNA (mRNA) and release of its mature protein. Lipopolysaccharide (LPS) was also found able to induce TNF-alpha secretion by PMN, but was a less potent stimulus compared with Y-IgG. There was no evidence of interleukin-6 (IL-6) gene expression in PMN after phagocytosis of Y-IgG or in response to LPS, whereas IL-6 mRNA expression and secretion were induced by either stimulus in monocytes. These findings demonstrate that a physiological function such as phagocytosis modulates the gene expression for a cytokine in PMN and shed new light on the understanding of the pathogenesis of the inflammatory process.

Impaired cytokine production by neutrophils isolated from patients with AIDS.

OBJECTIVES: To determine the ability of neutrophils isolated from HIV-seropositive patients to produce proinflammatory cytokines. DESIGN: The in vitro responsiveness of polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) to lipopolysaccharide (LPS), used in the presence or absence of interferon (IFN)gamma, was determined in 47 HIV-positive patients with advanced stages of virus infection. METHODS: Cytokines in cell-free supernatants were measured by enzyme-linked immunosorbent assay or radioimmunoassay. RESULTS: Cell-free supernatants from PMN isolated from the peripheral blood of HIV-positive patients and stimulated with LPS contained increased amounts of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 with respect to supernatants obtained from PMN of normal donors. In contrast, release of IL-1beta and IL-1ra (IL-1 receptor antagonist) in response to LPS, or LPS plus IFNgamma, was found to be lower in PMN from HIV-positive patients than in PMN from controls, but was significant only in the case of IL-1ra. Furthermore, the release of IL-12 induced by LPS or LPS plus IFNgamma did not significantly differ between PMN from HIV-positive patients and healthy donors. Concerning PBMC, the production of TNF-alpha and IL-12 in response to LPS, or LPS plus IFNgamma, was found to be significantly higher in cells isolated from HIV-positive patients, whereas the release of IL-1beta was significantly lower. In the case of IL-8, no statistically significant difference was found between PBMC isolated from HIV-positive patients and healthy donors. CONCLUSIONS: Collectively, the data suggest that in HIV-positive patients with advanced stages of disease, the ability of PMN to produce specific cytokines in response to LPS is significantly altered. Alterations in this ability might contribute to the evolution of HIV disease.

Activation of transcription factor NF-kappa B by phagocytic stimuli in human neutrophils.

Phagocytosis represents an important physiological trigger for the inducible expression of several genes in human neutrophils. Here, we report that a DNA-binding activity primarily consisting of the classical NF-kappa B heterodimer, p50/RelA, is induced in phagocytosing neutrophils. Under these conditions, NF-kappa B activation was found to be a rapid and transient response, reaching a maximum by 10-15 min, and returning to near-basal levels by 30 min. In neutrophils undergoing the phagocytosis of opsonized yeasts, the onset of NF-kappa B activation was paralleled by a decline in immunoreactive I kappa B-alpha protein levels, and the cellular I kappa B-alpha pool was replenished by 30 min, in agreement with our gel shift data. We conclude that NF-kappa B activation could constitute one of the mechanisms whereby the expression of kappa B-responsive genes is enhanced in phagocytosing neutrophils. To our knowledge, this represents the first demonstration that phagocytic stimuli can induce NF-kappa B activation in human neutrophils.

Granulocyte colony-stimulating factor induces the binding of STAT1 and STAT3 to the IFNgamma response region within the promoter of the Fc(gamma)RI/CD64 gene in human neutrophils.

Granulocyte colony-stimulating factor (G-CSF) has been recently shown to induce the high-affinity Fc receptor for IgG (Fc(gammaRI)/CD64) in human polymorphonuclear neutrophils (PMN). To elucidate the molecular mechanisms whereby G-CSF exerts this effect, we examined whether the cytokine induces the binding of transcription factors to the IFNgamma response region (GRR), a well characterized regulatory element in the Fc(gammaRI) promoter that is responsible for the transcriptional induction of this gene. Using electrophoretic mobility shift assays, we show that in human PMN, G-CSF activates a GRR-binding complex which contains members of the signal transducer and activator of transcription (STAT) family of proteins, namely STAT1 and STAT3. In keeping with this result, treatment of neutrophils with G-CSF led to tyrosine phosphorylation of STAT3, as determined by immunoprecipitation followed by immunoblotting with antiphosphotyrosine antibodies. This is the first demonstration that in human neutrophils, the induction by G-CSF of Fc(gammaRI) gene expression may be mediated by the binding of STAT1 and STAT3 to the GRR sequence.

Presence of cytochrome b-245 in NADPH oxidase preparations from human neutrophils.

The composition of NADPH oxidase purified by Red Sepharose chromatography of extracts from human neutrophil membranes was investigated. In contrast to that was recently reported by others, the enzyme isolated according to this procedure contained a high concentration of cytochrome b-245 and little FAD. The results reinforce the belief that cytochrome b-245 is a major component of the NADPH oxidase and plays a fundamental role in the formation of O2-by neutrophils.

On the detection of neutrophil-derived vascular endothelial growth factor (VEGF).

In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).

Measurement of NADPH oxidase activity in detergent lysates of human and mouse macrophage monolayers.

An assay to measure NADPH oxidase activity in detergent lysates of macrophage monolayers is described. The addition of a reaction mixture containing appropriate concentrations of disrupting detergents, NADPH as oxidase substrate and cytochrome c as electron acceptor, to macrophages monolayers permits the reliable detection of a superoxide dismutase-sensitive NADPH-dependent cytochrome c reductive activity. This activity is strictly substrate dependent and NADH could not substitute for NADPH. The NADPH-dependent superoxide anion-forming activity (NADPH oxidase) was investigated in different populations of human and mouse macrophages. NADPH oxidase was activated by stimulation of macrophages with phorbol-myristate acetate and activity levels correlated with ability of intact cells to produce superoxide anion. The optimal conditions for assay of NADPH oxidase were investigated and the assay was used to measure the kinetic properties of the NADPH oxidase. The assay permits investigations of the enzymatic basis of oxidative metabolism in macrophages cultivated as adherent cells without any requirements for recovery of the cells in suspension and subcellular fractionation.

Activation of nuclear factor-kappa B by beta-amyloid peptides and interferon-gamma in murine microglia.

An increasing body of evidence suggests that amyloid-beta (A beta) peptides and microglia are crucially involved in the pathogenesis of Alzheimer's disease. In an effort to further elucidate the biological effects of A beta towards microglia, we investigated the ability of A beta peptides to activate nuclear factor (NF)-kappa B in the N9 murine microglial cell line. Co-stimulation of microglia with suboptimal concentrations of A beta(25-35) and 100 U/ml IFN gamma resulted in the detection of a specific NF-kappa B DNA-binding activity in nuclear extracts, as determined in gel mobility shift assays. This response required at least 120 min to be evident and supershift experiments revealed that the NF-kappa B complex contains both RelA and p50. Accordingly, immunoblot experiments showed that amongst NF-kappa B/Rel proteins, RelA and p50 are mobilized to the nucleus following microglial cell stimulation with A beta(25-35) plus IFN gamma. Higher concentrations of A beta(25-35) were effective by themselves in inducing NF-kappa B activation, both in the N9 microglial cell line and in rat primary microglia, as well as in human monocytes. For purposes of comparison, microglia were also stimulated with bacterial LPS, a known NF-kappa B inducer. As expected, LPS strongly induced the formation of two NF-kappa B DNA-binding activities, one of which was identified as RelA/p50. The LPS response was also more rapid, as it was already evident by 40 min and remained sustained for up to 3 h. Collectively, these findings indicate that NF-kappa B activation might constitute one of the mechanisms underlying the inducible expression of kappa B-dependent genes in microglia stimulated by A beta peptides and IFN gamma, or by LPS.

beta-Amyloid(25-35) induces the production of interleukin-8 from human monocytes.

In an effort to unravel some of the cellular actions of beta-amyloid protein (A beta), we investigated its effects on interleukin-8 (IL-8) production from human monocytes. Supernatants harvested from cultured monocytes stimulated with the neurotoxic fragment 25-35 of beta-amyloid [A beta(25-35)] contained significant amounts of IL-8. Northern blot analysis demonstrated that A beta(25-35) also induced IL-8 mRNA accumulation. The effect of A beta(25-35) on IL-8 mRNA accumulation and secretion was not mimicked by a scrambled A beta(25-35) peptide, and was not affected by polymyxin B sulphate, which, on the other hand, almost completely abolished the effect of lipopolysaccharide. Our results uncover a new biological action of beta-amyloid: that of stimulating the production of a chemokine from monocytes.