Mononuclear cell adherence induces neutrophil chemotactic factor/interleukin-8 gene expression.

The accumulation of polymorphonuclear cells (PMN) in tissue is an essential element of the inflammatory response that is important in host defense. Adherence to endothelium constitutes the first step in PMN migration from the vascular compartment to the interstitium. We demonstrate that human peripheral blood mononuclear cells (PBMC) adherent to plastic can result in expression of interleukin-8 (IL-8), a potent PMN chemoattractant and activating cytokine. Northern blot analyses showed PBMC adherent to plastic expressed IL-8 steady-state mRNA levels by 30 min, peaked at 8 h, and then decreased over the next 16 h. In contrast, nonadherent PBMC (cultured in teflon chambers) expressed less than 25% of the maximal IL-8 steady-state mRNA levels as compared with adherent PBMC. Adherent PBMC-associated IL-8 determined by immunochemistry, supernatant chemotactic bioactivity, and extracellular antigenic IL-8 paralleled IL-8 mRNA expression. Antigenic and bioactive IL-8 were significantly apparent by 4-8 h, respectively, and increased significantly to maximal levels by 24 h. Furthermore, adherent PBMC IL-8 gene expression was suppressed by either concomitant treatment with actinomycin-D or cycloheximide, yet specific neutralizing antibodies directed against either IL-1 beta or tumor necrosis factor (TNF)-alpha failed to alter adherence-induced steady-state IL-8 mRNA levels. These data support the hypothesis that PBMC adherence is an important signal for the production of IL-8, and may be essential to the development of the inflammatory response through the elicitation of PMN.     

The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell-derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1beta was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-alpha, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-gamma down-regulated the GCP-2 production in fibroblasts induced by IL-1beta, TNF-alpha, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1beta, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte-derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.     

Control of chemokine production at the blood-retina barrier

Chemokine production at the blood-retina barrier probably plays a critical role in determining the influx of tissue-damaging cells from the circulation into the retina during inflammation. The blood-retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein-1 (MCP-1), regulated on activation of normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin (IL)-8, epithelial cell-derived neutrophil activating protein-78 (ENA-78) and growth related oncogene alpha (GROalpha) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP-1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL-1beta and tumour necrosis factor-alpha (TNF-alpha). MCP-1 and IL-8 were produced at much higher levels than the other chemokines tested. MIP-1alpha and MIP-1beta were present only at low levels, even after stimulation with IL-1beta and TNF-alpha. Cytokines with greater anti-inflammatory activity, such as IL-4, IL-10, IL-13, transforming growth factor-beta (TGF-beta) and IL-6, had little effect on chemokine production either by REC alone or after stimulation with IL-1beta and TNF-alpha. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site-specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.     

The Detection of a Novel Neutrophil-Activating Peptide (ENA-78) Using a Sensitive Elisa

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (<10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GROα; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.     

The detection of a novel neutrophil-activating peptide (ENA-78) using a sensitive ELISA.

Neutrophil elicitation into tissue is an essential element of the host defense in response to various stimuli, including, tissue injury, infection, or cancer. This event has gained renewed interest with the discovery of a family of small polypeptides (less than 10 kD). The salient features of these cytokines are the presence of four cysteine amino acids (first two separated by one amino acid; C-X-C) and their ability to induce neutrophil chemotaxis and activation. Recently, our laboratories have discovered a new member of this C-X-C chemotactic cytokine supergene family, neutrophil-activating peptide, ENA-78. ENA-78 shares significant amino acid sequence homology with neutrophil activating peptide-2 (NAP-2; 53%), growth regulated oncogene/melanoma growth stimulatory activity (GRO alpha; 52%), and IL-8 (22%). In addition, ENA-78 appears to activate neutrophils through the IL-8 receptor. Since both in vitro and in vivo biological fluids may contain an array of chemotactic cytokines that may be relevant to the activation and chemotaxis of neutrophils, we have developed a highly specific and sensitive sandwich ELISA for the detection of ENA-78.     

CXCR1-binding chemokines in inflammatory bowel diseases: down-regulated IL-8/CXCL8 production by leukocytes in Crohn's disease and selective GCP-2/CXCL6 expression in inflamed intestinal tissue

Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBD) that are characterized by chronic intestinal inflammation and a constant influx of leukocytes mediated by pro-inflammatory cytokines and chemokines. The intestinal expression of the CXCR1-binding chemokines IL-8/CXCL8 and GCP-2/CXCL6 and the participation of immunocompetent cells in IBD were evaluated. IL-8 production by peripheral blood mononuclear cells (PBMC) from IBD patients, stimulated with endotoxin, plant lectin or double-stranded RNA, was significantly lowered in patients with CD, but not in UC patients or healthy subjects. The reduced chemokine production by PBMC from IBD patients was both IL-8 and CD specific, but not inducer dependent. In serum, most chemokines remained undetectable, while the levels of those that were measurable remained unaltered in IBD patients. GCP-2, but not ENA-78/CXCL5, nor IL-8, were highly expressed by endothelial cells in inflamed intestinal tissue of IBD patients. In contrast, stimulated endothelial cell cultures produced more IL-8 than GCP-2. The selective GCP-2 staining of endothelial cells at sites of ulcerations suggests that GCP-2, despite its low production capacity in vitro, plays a role in IBD that is different from that of structurally (ENA-78) and functionally (IL-8) related ELR(+) CXC chemokines. Thus, the chemokine network shows complementarity rather than redundancy.     

Polymorphonuclear leucocytes selectively produce anti-inflammatory interleukin-1 receptor antagonist and chemokines, but fail to produce pro-inflammatory mediators

The role of neutrophils in the immune response has long been regarded as mainly phagocytic, but recent publications have indicated the production of several cytokines by polymorphonuclear leucocytes (PMN). The results of the individual reports, however, vary considerably. In this study, we established a cytokine profile of pure human neutrophils and demonstrated that minor contamination of peripheral blood mononuclear cells (PBMCs) in PMN preparations can lead to false-positive results. In our hands, peripheral blood PMN fail to produce the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNF-alpha). Instead, they secrete large amounts of the chemokine IL-8 and the anti-inflammatory IL-1 receptor antagonist (IL-1ra). Additionally, PMN preparations of a high purity show production of the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and growth-related oncogene-alpha (GRO-alpha), as well as macrophage colony-stimulating factor (M-CSF). The neutrophil therefore represents a novelty by producing the antagonist of IL-1beta (i.e. IL-1ra) in the absence of IL-1beta itself. To support our results, we differentiated stem cells from human cord blood into PMN and monocytes, respectively. These in vitro-differentiated PMN showed the same cytokine profile as peripheral blood PMN lacking IL-1beta, while differentiated monocytes produced the expected IL-1beta in addition to IL-1ra. The clear anti-inflammatory nature of their cytokine profile enables PMN to antagonize pro-inflammatory signals in experimental conditions. It is therefore possible that PMN play a key role in immune regulation by counteracting a dysregulation of the inflammatory process. Clinical studies, in which administration of recombinant G-CSF had a favourable effect on the outcome of severe infections and even sepsis without worsening inflammation, could thus be explained by our results.     

Activation of peripheral blood neutrophils from patients with active advanced tuberculosis.

Activation of peripheral blood neutrophils (PMN) was investigated in order to determine whether they might contribute to the inflammatory process during active advanced tuberculosis. Receptors for the Fc portion of IgG (FcgammaR) (FcgammaRI, FcgammaRII, and FcgammaRIIIB), CD66 (degranulation marker), and receptors for tumor necrosis factor-alpha (TNF-R55 and TNF-R75) were analyzed on PMN obtained from normal controls and tuberculosis patients (TB-PMN). Functional parameters such as cytotoxicity, superoxide anion generation triggered by N-formyl-methionyl-leucyl-phenyl-alanine (FMLP), and TNF-alpha and IL-1beta production were evaluated. A high expression of TNF-R55, CD66, and FcgammaRIIIB and the appearance of FcgammaRI were detected in TB-PMN. In addition, cytotoxicity, superoxide anion release, and TNF-alpha and IL-1beta production were enhanced in TB-PMN. Thus, in tuberculosis, the activation of PMN outside the focus of infection strongly suggests the possibility of a systemic inflammation that could modulate the inflammatory response.     

Tumor-derived granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor prolong the survival of neutrophils infiltrating bronchoalveolar subtype pulmonary adenocarcinoma

We evaluated the role of the tumor environment in the regulation of apoptosis of tumor-infiltrating neutrophils, the number of which correlates negatively with outcome, in patients with adenocarcinoma of the bronchioloalveolar (BAC) subtype. We examined three different parameters of apoptosis, namely morphological aspect, annexin-V expression, and DNA fragmentation. Bronchoalveolar lavage fluid (BALF) supernatants from patients with BAC significantly inhibited the 24-hour spontaneous apoptosis of normal peripheral blood neutrophils in vitro compared to BALF supernatants from control patients (64 +/- 4% versus 90 +/- 2% measured by annexin-V flow cytometry, P = 0.04). The alveolar neutrophil count correlated positively with the granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations in the patient's BALF. Furthermore, neutralizing antibodies (Abs) against GM-CSF and G-CSF significantly inhibited BALF anti-apoptotic activity (15 to 40% and 34 to 63% inhibition, respectively), whereas neutralizing Abs against interleukin (IL)-8, IL-6, IL-1beta and tumor necrosis factor-alpha had no significant effect. In an attempt to identify the cell origin of anti-apoptotic cytokines, we tested in vitro the effect of BAC cells (A549 cell line and primary culture derived from a patient's BAC tumor) on the apoptosis of peripheral blood neutrophils. Cell-free supernatants from tumor cells did not inhibit neutrophil apoptosis. In contrast, cell-free supernatants from tumor cells previously exposed to conditioned media from peripheral blood mononuclear cells and alveolar macrophages significantly inhibited spontaneous neutrophil apoptosis. This inhibition was partially lifted when conditioned media from mononuclear cells were previously treated with Abs against IL-1beta and tumor necrosis factor-alpha. As in vivo, neutralizing Abs against GM-CSF significantly inhibited the anti-apoptotic activity of cell culture supernatants, and combination with Abs against G-CSF had an additive effect. In vivo, GM-CSF and G-CSF were strongly expressed by tumor cells and moderately or not expressed by the normal epithelium, as assessed by immunohistochemical studies. These findings demonstrate that the tumor environment generates local conditions that prolong alveolar neutrophil survival through the production of soluble factors, thereby contributing to the persistence of the neutrophil alveolitis observed in BAC.     

Shiga Toxins Induce, Superinduce, and Stabilize a Variety of C-X-C Chemokine mRNAs in Intestinal Epithelial Cells, Resulting in Increased Chemokine Expression

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.     

Role of neutrophils in release of some cytokines and their soluble receptors

Available data suggest that cytokines and soluble cytokine receptors, which are being produced by immunological cells, can modulate the immune response of the host. Although the production of mediators such as TNF-alpha and IL-6 as well as that of their soluble receptors has been extensively studied in tissue cells and mononuclear cells, it has not been fully investigated in neutrophils (PMN). In the present study we examined the ability of PMN to simultaneously release TNF-alpha, IL-6 and their soluble receptors-sTNFRp55, sTNFRp75 and sIL-6R. Concentrations of soluble receptors were compared with expression of membrane-bound TNF and IL-6 receptors. For comparative purposes, similar examinations with autologous peripheral blood mononuclear cells (PBMC) were performed. We found that PMN and PBMC have the same ability to release IL-6 and sTNFRp75. In contrast, there were significant differences in the release of TNF-alpha, sTNFRp55 and sIL-6R between these cells. Reduction in membrane TNF receptor expression, observed in this study, was associated with increase secretion of soluble TNF receptors by PMN and PBMC. The results suggest that PMN can play an essential role in modulating the inflammatory response by affecting the balance between pro-inflammatory mediators, such as TNF-alpha, and anti-inflammatory mediators, such as soluble TNF receptors.     

Expression of IL-6 and TNF-alpha in human alveolar epithelial cells is induced by invading, but not by adhering, Legionella pneumophila

Legionella pnemophila causes atypical pneumonia in humans, especially in patients with chronic pulmonary diseases and underlying immunosuppression, and in elderly people. Several previous studies have shown that L. pneumophila induced several inflammatory cytokines in murine macrophages, but little is known about cytokine induction by the bacterium in lung epithelial cells. In this study, we investigated the ability of L. pneumophila to stimulate the production of pro-inflammatory cytokines in the human A549 alveolar epithelial cell line during 24h exposure to 10(6), 10(7), and 10(8) microbes. Infection of the wild L. pneumophila strain to A549 resulted in increased levels of interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-alpha) mRNA, and also the secretion of their production into culture medium. In contrast, the level of mRNAs and proteins of IL-1beta and gamma interferon (IFN-gamma) remained unchanged and undetected, respectively. Production of IL-8, IL-6, and TNF-alpha in A549 decreased when an icmE multiplication-less mutant and the heat-killed L. pneumophila strain were inoculated. The treatment of cytochalasin D, which effectively inhibited invasion of L. pneumophila into A549, significantly reduced the production of IL-6 and TNF-alpha, but not IL-8. These results suggested that the induction and expression of IL-6 and TNF-alpha in the human alveolar epithelial cells especially required intracellular signaling by L. pneumophila after invasion.     

Novel CD8 molecule on macrophages and mast cells: expression, function and signaling

BACKGROUND: We previously identified, using flow cytometry and in situ RT-PCR, a novel CD8 molecule on rat alveolar macrophages (AM) and mast cells (MC). RT-PCR also demonstrated that mouse AM express CD8 mRNA. Functional studies on rat AM determined that ligation of CD8 alpha- and beta-chains induced inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO), TNF-alpha and IL-1beta (CD8alpha only) secretion. However, CD8 did not induce AM phagocytosis of IgG-opsonized or unopsonized particles. Rat MC stimulated through CD8 secreted NO and TNF-alpha, but not histamine. Because of its potential role in regulating cell function, we investigated the signaling pathways involved in macrophage CD8 stimulation. METHODS AND RESULTS: Inhibitor of src family kinases (PP1) significantly (p<0.05) inhibited CD8alpha (OX8 antibody)-induced iNOS upregulation, NO, TNF-alpha and IL-1beta production in rat AM. In addition, Ro 31-8220 (a PKC inhibitor) inhibited OX8-induced iNOS upregulation, NO and IL-1beta production, but did not inhibit TNF-alpha production. Using Syk antisense, we further determined that OX8 stimulation of NO is Syk kinase dependent. CONCLUSION: Studies on the signaling mechanisms of CD8 determined that src family kinases, PKC, and Syk kinase are involved in CD8 signaling. Additionally, CD8 may have differential signaling pathways, as an inhibitor to PKC downregulated OX8-induced IL-1beta, but not TNF-alpha release. Our studies demonstrate that AM CD8 is similar to T lymphocyte CD8 in that src kinases are involved in CD8-mediated signaling. However, p56(lck), which is expressed in T lymphocytes, has not been found in macrophages, suggesting that other src family kinases may be involved in AM and MC CD8 signaling.     

Neutrophils isolated from leprosy patients release TNF-alpha and exhibit accelerated apoptosis in vitro

This study demonstrated that polymorphonuclear neutrophils (PMN) participate in the acute inflammatory response in leprosy as effector cells. Lepromatous patients present intense infiltrate of neutrophils in reactional (ENL) lesions. Circulating PMN of nonreactional patients, healthy donors, and reactional patients were purified and analyzed in vitro. The study confirmed the short lifespan of these cells in culture with progressive changes characteristic of apoptosis. Apoptosis was greatly accelerated in ENL patients as shown by cellular morphology, later confirmed by qualitative and quantitative analysis of fragmented DNA. It was observed that neutrophils stimulated with lipopolysaccharide, Mycobacterium leprae, and lipoarabinomannan secrete interleukin-8 and tumor necrosis factor alpha (TNF-alpha). Thalidomide, a drug known to inhibit TNF-alpha synthesis on monocytes, also exerted an inhibitory effect on TNF-alpha secretion in neutrophils. These data suggest that PMN can participate in the regulation of the immune response in leprosy and can contribute to the amplification of TNF-alpha production at the site of ENL lesion.