Production Of Neutrophil Chemotactic Factors Research Articles

Interleukin(IL)-4 promotion of CXCL-8 gene transcription is mediated by ERK1/2 pathway in human pulmonary artery endothelial cells

Interleukin-4 is central to allergic pulmonary inflammatory responses, but its contribution to airway neutrophilia remains controversial. The endothelium plays a critical role in regulating leukocyte recruitment and migration during inflammation. However, its response to IL-4 is reported to either increase or decrease the production of neutrophil chemotactic factors. We hypothesized that these conflicting findings may be due to the tissue and the size of the vessels from which endothelial cells have been derived. The expression of CXCL-8 by human primary culture umbilical veins endothelial cells (HUVECs), human pulmonary artery endothelial cells (HPAECs), and human pulmonary microvascular endothelial cells (HPMECs) when stimulated with recombinant human IL-4 (rhIL-4) was studied. The chemoattractant property of the cells’ supernatants for neutrophils was evaluated using Boyden chambers. The role of the nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPK) in IL-4-induced HPAECs was studied using Western blotting and electrophoretic mobility shift assay (EMSA). We demonstrated that IL-4 increased the mRNA expression and the protein production of CXCL-8 in HPAECs, but not in HUVECs and HPMECs. The supernatants of HAPECs stimulated by IL-4 significantly promoted neutrophils migration in a dose-dependent manner, and was significantly attenuated by an inhibitor of CXCL-8. We also found that extracellular-regulated protein kinase1/2 (ERK1/2) is activated by IL-4 in HPAECs, but not JUN-N-terminal protein kinase (JNK) or p38 MAPK pathway. Furthermore, NF-κB–DNA binding activity, phosphorylation of IκBα and p65 levels were not affected by rhIL-4 in HAPECs. These findings indicate marked functional differences in the response of micro and macro-ECs to IL-4. ERK1/2, rather than NF-κB, JNK and p38 MAPK signaling, plays a role in IL-4 induced chemokine activation. Our results suggest that inhibition of ERK1/2 may be a possible target for airway neutrophilia in allergic lung diseases.

Neutrophil chemotactic factor in supernatant from pulmonary fibroblasts stimulated with cytokines

Fibroblasts are important for maintenance of the structural frame network for most tissues, and they also play an important role in the inflammatory process via production of various mediators. In this study, we demonstrated that pulmonary fibroblasts may participate in pulmonary inflammation by production of neutrophil chemotactic factor (NCF). Pulmonary fibroblasts were stimulated with various cytokines (IGF-1, PDGF, IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF, IFNr). Fibroblasts stimulated with either TNF, IL-1 alpha or IL-beta but not IGF, PDGF, IL-2 or IL-6 demonstrated a kinetic and dose-dependent increase in NCF activity. The NCF activity of crude supernatant was heat-stable and was not changed by anti-C5 antibody treatment or ether extraction. Characterization of the NCF activity by gel-filtration using high pressure liquid chromatography showed two active fractions, one with MW greater than 100 kD and the other with MW less than 10 kD. NCF activity in the small molecular weight fraction was demonstrated by inhibition of chemotaxis by addition of anti-IL-8 antibody. These data suggest that cytokine-treated fibroblast-derived NCF may be important in the pathogenesis and expression of a variety of pulmonary disease processes associated with neutrophil accumulation and activation.

In Vitro Exposure To Cigarette Smoke Activates Eosinophils: Implications For Lung Inflammation

Effects of cigarette smoke (CS) on eosinophils (EOS), important cells involved in the pathogenesis of chronic lung diseases such as asthma, were studied in vitro. EOS were isolated from healthy and mildly atopic donors and exposed to soluble components of cigarette smoke (CSE). Viability and apoptosis were assessed by flow cytometry after staining with propidium iodide and Annexin-V. Activation was determined by release of newly-synthesized (IL-8, IL-6) mediators and by phosphorylation of MAPKs. CSE effects on ultrastructural morphology and production of neutrophil chemotactic factors in CSE-activated EOS were also evaluated. CSE concentrations from 0-2.5% were non-toxic for up to 18-24 hours of exposure. However, CSE at 2.5% activated EOS as evidenced by ultrastructural degranulation: release of IL-8 and IL-6, and increased expression of the MAPK, c-Jun. Supernatants from CSE-activated EOS were found to be significantly chemotactic for neutrophils. These results suggest that CS may aggravate lung inflammation by activating EOS which, in turn, release inflammatory mediators promoting inflammatory cell recruitment and lung remodeling.

Anti-inflammatory effects of Erythromycin and Tetracycline on Propionibacterium. acnes induced production of chemotactic factors and reactive oxygen species by human neutrophils

Propionibacterium acnes (P. acnes), an anaerobic pathogen, plays an important role in the pathogenesis of acne and seems to initiate the inflammatory process by producing neutrophil chemotactic factors (NCF). Once neutrophils attracted by bacterial chemoattractants reach the inflamed site, they release inflammatory mediators such as lysosomal enzymes and reactive oxygen species (ROS). Previously, it has been shown that antibiotics may affect acne by means other than their anti-bacterial effects. Thus, we investigated the effect of subminimal inhibitory concentration (sub-MIC) of tetracycline and erythromycin on production of NCF and ROS. NCF was tested in vivo in a mouse model and ROS was estimated on human PMNL in vitro, by nitroblue tetrazolium dye reduction test (NBT) and cytochrome-C reduction test. Tetracycline (CS-T) and Erythromycin (CS-E) treated cultures showed a significant reduction of 35.8% and 58.3% in NCF production respectively, as compared to P. acnes stimulated cultures. Tetracycline and erythromycin at their sub-MIC also significantly inhibited release of ROS from human PMNL. Thus, tetracycline and erythromycin, besides having antibacterial activity, also have an anti-inflammatory action. These antibiotics reduce the capacity of P. acnes to produce NCF, as well decrease its ability to induce ROS from PMNL.

Effects of Roxithromycin on the Production of Lipase and Neutrophil Chemotactic Factor by Propionibacterium acnes

Background: The macrolide antibiotic roxithromycin is effective against acne associated with inflammation, but the mechanism by which this is achieved has not been clarified. Objective: We studied the effects of roxithromycin on the production of lipase and neutrophil chemotactic factor by Propionibacterium acnes in vitro. Results: Roxithromycin significantly inhibited the production of lipase and neutrophil chemotactic factor by P. acnes at a concentration one eighth of the MIC, at which the growth curve of P. acnes is not affected. Conclusion: One mechanism of the effectiveness of roxithromycin in acne therapy is thought to be the inhibition of bacterial lipase and neutrophil chemotactic factor production by P. acnes.

Effect of parainfluenza-3 virus challenge on cell-mediated immune function in parainfluenza-3 vaccinated and non-vaccinated calves

A group of four conventional, colostrum-fed calves was vaccinated with live parainfluenza type 3 (PI-3) virus vaccine at 1 and 5 weeks of age. A group of four control calves was treated with cell culture medium at the same time. Two weeks after the second vaccination, both groups of calves were challenged with PI-3 virus by a combined respiratory route. Blood and nasal mucus samples were collected at intervals, and alveolar macrophages were recovered before and after challenge by bronchoalveolar lavage. The results demonstrated that clearance of virus, as indicated by presence of virus antigen was more rapid in previously vaccinated calves. Several alveolar macrophage functions were markedly reduced in all calves 5 to 7 days following virus challenge, although microbicidal activity was unaffected, compared to the controls. The production of neutrophil chemotactic factors by alveolar macrophages occurred more rapidly after virus challenge in the previously vaccinated calves and this correlated with a more rapid neutrophil influx into the lungs in these animals.

Induction of neutrophil chemotactic factor production by staurosporine in rat peritoneal neutrophils

1. Incubation of rat peritoneal neutrophils in medium containing various concentrations of staurosporine (6.4-64 nM) increased the neutrophil chemotactic activity in the conditioned medium in a time- and concentration-dependent manner. 2. Separation of the neutrophil chemotactic activity in the conditioned medium by isoelectric focusing revealed that staurosporine (64 nM) stimulated the production of basic (pH > 8) neutrophil chemotactic factors, while TPA (12-O-tetradecanoylphorbol 13-acetate, 49 nM) stimulated the production of both basic (pH > 8) and acidic (pH 5) neutrophil chemotactic factors. 3. Determination by immunoassay of cytokine-induced neutrophil chemoattractant (CINC)-1, -2 alpha, -2 beta and -3 in the conditioned medium at 4 h revealed that staurosporine (64 nM) and TPA (49 nM) strongly stimulated the production of CINC-3 (staurosporine, 133.0 +/- 3.8; TPA, 26.7 +/- 1.0; control, 0.32 +/- 0.01 ng ml-1, means +/- s.e.mean from four samples) compared to CINC-1 (staurosporine, 55.0 +/- 1.2; TPA, 12.2 +/- 0.3; control, 0.56 +/- 0.01 ng ml-1), and CINC-2 alpha (staurosporine, 1.09 +/- 0.03; TPA, 0.90 +/- 0.02; control, < 0.10 ng ml-1). CINC-2 beta was below the detectable amount (< 0.078 ng ml-1). 4. The level of CINC-3 mRNA in the peritoneal neutrophils was determined by reverse transcription-polymerase chain reaction. Staurosporine (64 nM) and TPA (49 nM) enhanced the level of CINC-3 mRNA time-dependently, but had no effect on GAPDH mRNA levels. 5. Production of staurosporine-induced neutrophil chemotactic factor was inhibited by the protein kinase C inhibitors, H-7 (IC50, 12.3 microM), calphostin C (IC50, 0.77 microM) and Ro 31-8425 (24.3% inhibition at 10 microM), and by the tyrosine kinase inhibitor, genistein (IC50, 68.5 microM). Production of TPA-induced neutrophil chemotactic factor was also inhibited by both inhibitors. 6. Both the staurosporine- and the TPA-induced increase in CINC-3 mRNA levels were suppressed by H-7 and genistein.

Effect of BVD virus infection on alveolar macrophage functions

Alveolar macrophages (AM) were recovered by bronchoalveolar lavage from a group of eight calves at various times before and after inoculation with a cytopathic respiratory isolate of bovine viral diarrhoea virus (BVDV). A second group of four calves were given tissue culture medium as a control inoculum. Macrophages were also recovered from two additional, uninoculated calves, and were exposed to BVDV in vitro. Tests were carried out on the recovered macrophages to determine the effects of the virus on several functional properties. Immunofluorescence did not indicate the AM as being readily susceptible to this isolate of BVDV, although infection did occur. Fc receptor (FcR) and complement receptor (C3R) expression, phagocytosis and microbicidal activity and the production of neutrophil chemotactic factors were all significantly reduced in macrophages recovered from BVDV infected calves, compared with pre-inoculation control levels, whereas the control inoculated calves displayed significant increases in some of the functions. With macrophages exposed to the virus in vitro however, only FcR and C3R expression and phagocytic activity were significantly reduced. The results demonstrate that BVDV can reduce local immune defences in the lung, following infection by the respiratory route, and in conjunction with the other immunosuppressive properties of BVDV would favour a pre-disposing role for the virus in the pathogenesis of respiratory disease in calves.

OK-432 induces production of neutrophil chemotactic factors in malignant pleural effusion.

We investigated the changes in cellular components and neutrophil chemotactic factors in pleural fluid from 19 lung cancer patients who received intrapleural injection of OK-432 to treat malignant pleurisy. Not only neutrophil chemotactic activity (NCA) but also neutrophil count and percentage were increased significantly at 6 hours after OK-432 injection. The neutrophil count was significantly correlated with NCA level. The levels of C5a and IL-8 in pleural fluid were increased significantly after OK-432 injection. The increased IL-8 level was associated with a increase of both NCA and neutrophil count. OK-432 treatment also induced a marked increase of IL-1 beta and IL-6 in pleural fluid. Thus, intrapleural injection of OK-432 induced production of neutrophil chemotactic factors (IL-8 and C5a) and cytokines (IL-1 beta and IL-6), which eventually attracted neutrophils into the pleural space. These observations suggest that neutrophil migration mediated by these factors and cytokines may contribute to the sclerosing effects of OK-432 treatment.

アレルギー性炎症における浸潤白血球による好中球走化性因子の産生機序とその実体

アレルギー性炎症における浸潤白血球による好中球走化性因子の産生機序とその実体

Possible roles of protein kinases in neutrophil chemotactic factor production by leucocytes in allergic inflammation in rats.

1. In an air pouch-type allergic inflammation model in rats, leucocytes that had infiltrated into the pouch fluid collected 4 h after the antigen challenge produced proteinaceous chemotactic factors for neutrophils when they were incubated in the medium. 2. To clarify the mechanism of activation of the infiltrated leucocytes in producing these factors, the effects of protein kinase inhibitors on neutrophil chemotactic factor production were examined. 3. When the infiltrated leucocytes were incubated for 4 h in medium containing the non-selective protein kinase inhibitor K-252a (1-100 ng ml-1, 2.14-214 nM), the tyrosine kinase inhibitor genistein (1-50 micrograms ml-1, 3.7-185 microM), and the more selective protein kinase C inhibitor H-7 (5-100 micrograms ml-1, 13.7-274 microM); neutrophil chemotactic activity in the conditioned medium was decreased in a concentration-dependent manner, but the adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor H-89 (1-1000 ng ml-1, 2.24-2240 nM) showed no effect. 4. Isoelectric focusing of the conditioned medium revealed that the leucocytes produced two neutrophil chemotactic factors, leucocyte-derived neutrophil chemotactic factor (LDNCF) 1 and LDNCF-2. Treatment of the leucocytes with K-252a, genistein, and H-7, but not H-89, inhibited production of both LDNCF-1 and LDNCF-2. 5. These results suggest that activation of tyrosine kinase and protein kinase C, but not cAMP-dependent protein kinase, is responsible for the production of LDNCF-1 and LDNCF-2. 6. The steroidal anti-inflammatory drug dexamethasone and the protein synthesis inhibitor cycloheximide inhibited neutrophil chemotactic factor production in a concentration-dependent manner. Time-course experiments showed that the inhibitory effect by dexamethasone was apparent even 30 min after the incubation.7. Mechanism for inhibiting the production of LDNCF-1 and LDNCF-2 by dexamethasone is also discussed.

Pseudomonas-induced neutrophil recruitment in the dog airway in vivo is mediated in part by IL-8 and inhibited by a leumedin

A bacteria-free supernatant of Pseudomonas aeruginosa induces the production of neutrophil chemotactic activity in human bronchial epithelial cells in vitro that is due to the potent chemotactic factor, interleukin-8 (IL-8). Because P. aeruginosa supernatant itself is not chemotactic, we hypothesized that intratracheal P. aeruginosa induces the production of neutrophil chemotactic factors, including IL-8, in vivo. Because neutrophils play a key role in cystic fibrosis, inhibition of neutrophil recruitment might be therapeutic. We studied the effect of P. aeruginosa supernatant in the isolated tracheal segment of dogs in vivo, and we measured neutrophil chemotactic activity in vitro in the tracheal fluid. We also determined the local effect of intratracheal administration of leumedin NPC 15669, an inhibitor of neutrophil recruitment, on IL-8- and Pseudomonas-induced neutrophil accumulation. P. aeruginosa supernatant and IL-8 both caused time-dependent accumulation of neutrophils in the tracheal fluid. Tracheal fluid obtained after P. aeruginosa administration had neutrophil chemotactic activity in vitro that was significantly inhibited by the IL-8 antibody. Intratracheal NPC 15669 prevented both IL-8- and Pseudomonas-induced accumulation of neutrophils. We conclude that P. aeruginosa supernatant recruits neutrophils into the airway indirectly by inducing the production of chemotactic factors, including IL-8. Our results suggest a potential therapeutic role for leumedins in chronic airway diseases.

P-499 - Effects of protein kinase inhibitors on neutrophil chemotactic factor production by leukocytes infiltrated into inflammatory sites.

P-499 - Effects of protein kinase inhibitors on neutrophil chemotactic factor production by leukocytes infiltrated into inflammatory sites.

Pharmacological analysis of neutrophil chemotactic factor production by leucocytes and roles of PAF in allergic inflammation in rats.

1. The role of platelet activating factor (PAF) in neutrophil infiltration in air pouch type allergic inflammation in rats was investigated. 2. Neutrophil infiltration into the pouch fluid 8 h after injection of the antigen (azobenzenearsonate-conjugated acetyl bovine serum albumin) solution into the air pouch of immunized rats was inhibited dose-dependently by treatment with PAF antagonists (CV-3988, L-652,731 and Y-24,180) in parallel with the decrease in neutrophil chemotactic activity in the pouch fluid. 3. Four hours after injection of the antigen solution into the air pouch of immunized and non-immunized rats, there was no significant difference between the two groups in the number of total leucocytes, neutrophils, mononuclear cells and eosinophils in the pouch fluid. However, when the infiltrated leucocytes were incubated in medium, chemotactic factor production by leucocytes from immunized rats was greater than that from non-immunized rats. 4. When leucocytes from non-immunized rats were preincubated for various periods in the medium containing 10 or 50 nM of PAF, washed, and further incubated in the medium containing no PAF, chemotactic factor production was not stimulated. 5. The increase in the chemotactic activity in the conditioned medium was not suppressed by the 5-lipoxygenase inhibitor, AA861. In addition, the chemotactic activity in the conditioned medium was not inhibited by the PAF antagonists. 6. Incubation of the infiltrated leucocytes in the medium containing the protein synthesis inhibitor, cycloheximide, inhibited chemotactic factor production in a concentration-dependent manner in parallel with the decrease in uptake of [3H]-leucine into the acid-insoluble fraction of leucocytes. 7. Separation of the chemotactic activity in the conditioned medium by isoelectric focusing revealed that the leucocyte infiltrated into the pouch fluid produce two kinds of factors, viz. leucocyte-derived neutrophil chemotactic factor-i (LDNCF-1) and LDNCF-2 of which pI values are 4-5 and above 8,respectively.8. The results indicate that PAF has no significant role in leucocyte activation to produce chemotactic factors, and that neutrophil chemotactic factors produced by infiltrated leucocytes are not PAF or leukotriene B4 but are produced through a protein synthesis mechanism.9. The mechanism of action of PAF antagonists on neutrophil infiltration into the inflammatory locus is discussed.

Production of Cytokines Belonging to the Interleukin-8 Family by Human Gingival Fibroblasts Stimulated with Interleukin-1β in Culture

Three major neutrophil chemotactic peptides belonging to the interleukin-8 (IL-8) family were purified to homogeneity from the conditioned medium of the IL-1β-stimulated human gingival fibroblasts culture. On the basis of their molecular masses and NH 2-terminal amino acid sequences, these three neutrophil chemotactic factors were identical to melanoma growth stimulatory activity-α (MGSA/ gro-α), MGSA/ gro-γ and Ala-Val-Leu-Pro-Arg-IL-8 (AVLPR-IL-8), each belonging to the IL-8 family. It has been shown by the chemotaxis studies in vitro that the chemotactic activity of MCSA/ gro-γ is similar to that of MGSA/ gro-α for both human and rat neutrophils, while AVLPR-IL-8 is chemotactic for human neutrophils but not for rat neutrophils. The in vitro findings were supported by the histological studies in vivo on the intradermal injection of either MGSA/ gro-α or AVLPR-IL-8 into the back of the rats. The results obtained in the present experiment suggest that human gingival fibroblasts play an important role in gingival inflammation through the production of neutrophil chemotactic factors newly characterized as the IL-8 family in response to inflammatory chemical mediators including IL-1β.

Effects of two adenoviruses (type 1 and type 8) on functional properties of bovine alveolar macrophages in vitro

Summary Cultures of bovine alveolar macrophages were inoculated with type-1 and type-8 adenoviruses, initially isolated from calves with respiratory tract disease, and functional properties of the cells were observed over a period of 10 to 11 days. Both viruses replicated in macrophages; viral titers were low (&lt; 3.75 log10 tcid5O/0.1 ml), and intranuclear inclusions were detected by indirect immunofluorescence in 5 to 10% of the cells from 3 days after inoculation. Highest titers were induced by type-1 adenovirus, which also induced the greatest functional changes. Expression of Fc and complement receptors was reduced by both viruses, although the greatest effects were seen with type 1. Phagocytosis of Candida krusei cells was reduced following type 1 infection, whereas phagocytosis in type-8-infected cells was not different from that of noninfected macrophages. Ability to kill ingested Candida cells also was reduced following type-1 infection, whereas type-8-infected macrophages had lower killing ability only at 2 to 4 days after inoculation. Neither virus had substantial effects on the production of neutrophil chemotactic factors by the macrophages.

ざそう病巣部細菌による好中球遊走化因子産生に対するｃｌｉｎｄａｍｙｃｉｎの最小発育阻止濃度以下での影響

The effect of clindamycin (CLDM) at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, Propionibacte-rium acnes (P. acnes) strains and coagulase-negative staphylococci (CNS) strains, was assayed using Boyden chamber method. Sub-MIC of CLDM markedly inhibited the production of neutrophil chemotactic factors in all strains of P. acnes and CNS. Our results seem to suggest that sub-MIC of CLDM has an anti-inflammatory effect presumably due to the inhibition of neutrophil chemotactic factors production in comedonal bacteria on the inflammatory process of acne.

Inhibition of neutrophil chemotactic factor production in comedonal bacteria by subminimal inhibitory concentrations of erythromycin.

The effect of 1/10 minimal inhibitory concentrations (sub-MIC) of erythromycin (EM) on human neutrophil chemotactic factor production in comedonal bacteria, Propionibacterium acnes strains, Propionibacterium granulosum strains and coagulase-negative staphylococcus (CNS) strains was assayed using the Boyden chamber method. Sub-MIC of EM significantly suppressed neutrophil chemotactic factor production in all strains of P. acnes, P. granulosum and CNS. Our results suggest that sub-MIC of EM may have an anti-inflammatory action by reducing the inflammatory capacity of comedonal bacteria in inflammatory acne.

Effect of “in vitro” exposure of bovine alveolar macrophages to different strains of bovine respiratory syncytial virus

A vaccine strain of respiratory syncytial (RS) virus and an isolate from pneumonic calves (AC2) were inoculated onto cultures of bovine alveolar macrophages recovered by lung lavage, and the functional properties of the cells observed over a period of 10 days. In most cultures no infectious virus was produced although immunofluorescence indicated the presence of virus antigens in some cells. No significant difference was noted between infected and control macrophage cultures in their capacity to phagocytose latex particles (neutral phagocytosis), although the ability to phagocytose complement-coated Candida krusei cells was affected, particularly with the AC2 strain after 6 days. Killing of C. krusei cells was slightly affected by infection of macrophages with the vaccine strain and was dramatically affected by infection with strain AC2. C3b and Fc receptor expression was adversely affected by both virus strains. Production of neutrophil chemotactic factors was increased in cultures infected with both strains, but was greater with AC2, suggesting that some properties of the cells were activated.

O-202 - Pharmacological analysis of neutrophil chemotactic factor production by infiltrated leukocytes in allergic inflammation in rats

O-202 - Pharmacological analysis of neutrophil chemotactic factor production by infiltrated leukocytes in allergic inflammation in rats