Smoke Extract Stimulates Lung Epithelial Cells to Release Neutrophil and Monocyte Chemotactic Activity

Inflammatory cells accumulate within the lungs of cigarette smokers. Current concepts suggest that these cells can induce protease-antiprotease and/or oxidant-antioxidant imbalance(s), which may damage the normal lung alveolar and interstitial structures. Because type II pneumocytes line the alveolar space, and because the inflammatory cells migrate and reside at the alveolus, we postulated that the type II pneumocytes might release chemotactic activity for neutrophils and monocytes in response to smoke extract. To test this hypothesis, A549 cells were cultured and the supernatant fluids were evaluated for the neutrophil and monocyte chemotactic activity (NCA and MCA) by a blind-well chamber technique. A549 cells released NCA and MCA in response to smoke extract in a dose- and time-dependent manner (P < 0.05). Checkerboard analysis showed that the activity was chemotactic. Partial characterization of NCA and MCA revealed that the activity was partly heat labile, trypsin sensitive, and ethyl acetate extractable. Lipoxygenase inhibitors and cycloheximide inhibited the release of NCA and MCA. Molecular sieve column chromatography showed multiple peaks for both NCA and MCA. NCA was inhibited by anti-human-interleukin (IL)-8 antibody, granulocyte colony-stimulating factor (G-CSF) antibody, or leukotriene (LT)B₄ receptor antagonist. Monocyte chemoattractant protein (MCP)-1 antibody or LTB₄ receptor antagonist inhibited MCA. Immunoreactive IL-8, G-CSF, MCP-1, and LTB₄ significantly increased in the supernatant fluids in response to smoke extract. These data suggest that the type II pneumocytes may release NCA and MCA and modulate the inflammatory cell recruitment into the lung.     

Generation of Heat-Labile Chemotactic Activity in Blood after Inhalation Challenge and its Relationship to Neutrophil and Monocyte/Macrophage Turnover and Activity

The chemotactic activity of serum has been monitored in eight patients with bronchial asthma after inhalation of an allergen. Lactoferrin and lysozyme were measured in serum simultaneously as markers of neutrophil and monocyte/macrophage activity and turnover. The heat-labile chemotactic activity of serum showed a two-phase response with an initial reduction in activity (median = 37.5 min after challenge) ( P < 0.05) followed by an increased activity (median = 60 min after challenge) ( P < 0.01). The consistency of this pattern was highly significant ( P < 0.0005). Heat-stable chemotactic activity was significantly increased ( P < 0.01) 15 min after challenge.
The initial reduction in heat-labile chemotactic activity was observed only in those patients who developed a late asthmatic response and could suggest the involvement of immune complex-mediated reactions since a similar reduction in heat-labile chemotactic activity is produced by in vitro activation of serum with aggregated IgG.
The increased heat-labile chemotactic activity showed a close relationship ( P < 0.01) to the extent of the immediate reduction in PEF-rate. No relationship was discernible between the chemotactic activity and the eventually occurring rise in PMN blood count. In contrast the activity and turnover of PMNs after challenge as reflected by serum-lactoferrin levels were significantly reduced ( P < 0.01). A close correlation ( P < 0.001) was found between the heat-labile chemotactic activity in serum and the serum-levels of lysozyme, suggesting the involvement of macrophages/monocytes in the asthmatic reaction and in the generation of the heat-labile chemotactic activity found in serum after inhalation challenge.     

Helicobacter pylori Lipopolysaccharide Binds to CD14 and Stimulates Release of Interleukin-8, Epithelial Neutrophil-Activating Peptide 78, and Monocyte Chemotactic Protein 1 by Human Monocytes

Helicobacter pylori gastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whether H. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whether H. pylori lipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to an H. pylori water extract (HPE) or to purified H. pylori LPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution of H. pylori LPS to monocyte activation was determined by using the LPS antagonist Rhodobacter sphaeroides lipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. Purified H. pylori LPS also stimulated monocyte chemokine production but was 1,000-fold less potent than Salmonella minnesota lipid A. RSLA blocked H. pylori LPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%, P < 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%, P < 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%, P < 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS. H. pylori LPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency, H. pylori LPS may play an important role in the pathogenesis of H. pylori gastritis.     

Increased Neutrophil Chemotactic Activity in Exercise- and "Fog"-Induced Asthma

To investigate whether exercise- and ultrasonic "fog"-induced asthma are due to the same mechanism, i.e. mediator release induced by osmotic changes, we measured the serum neutrophil chemotactic activity before and after exercise and inhalation of "fog" in 15 asthmatic subjects. To assess changes in airway caliber we measured specific airway conductance (SGaw); to assess changes in neutrophil chemotactic activity we measured the maximum distance reached by neutrophils in a filter when challenged with the subject's serum in a Boyden chamber. In 10 subjects, SGaw decreased by more than 35% and neutrophil chemotactic activity increased significantly (P less than 0.05) both after exercise and "fog", whereas in five subjects no change occurred either after exercise or "fog". We conclude that both exercise- and "fog"-induced asthma are associated with increased serum neutrophil chemotactic activity, and that both stimuli may cause asthma by osmotically triggering mediator release from mast cells.     

Monocyte and Neutrophil Adhesion to Matrix Proteins is Selectively Enhanced in the Presence of Inflammatory Mediators

The authors investigated the time course of monocyte and neutrophil adhesion to fibronectin, vitronectin and albumin precoated culture wells, using mixed leucocyte populations from healthy blood donors. Moreover, the influence of chemotactic agonists on the adhesion properties as well as the quantitative expression of CD29, CD11b/CD18 and CD61 was analysed by flow cytometry. Different chemotactic agonists were used representing a classical chemotactic agonist (fMLP), and agonists with a preferential effect on monocytes (RANTES) and neutrophils (IL-8), respectively. The authors found a gradual increase in monocyte and neutrophil adhesion to all three surfaces, reaching a plateau at 15 min of incubation. Adhesion to fibronectin was significantly higher at all time points (5, 15 and 60 min, respectively) compared with vitronectin and albumin in both monocytes and neutrophils. Neutrophil adhesion to vitronectin was significantly lower at 60 min compared with 15 min. Monocyte adhesion to albumin was increased by fMLP and RANTES and to vitronectin also by IL-8. Neutrophil adhesion to albumin and vitronectin was increased by fMLP and IL-8, but not RANTES. The adhesion to fibronectin was not altered by any of the chemotactic agonists used. The quantitative levels of CD11b/CD18, but not CD29 and CD61, was increased by fMLP, but not RANTES nor IL-8. The authors conclude that the adhesion of human monocytes and neutrophils to vitronectin and albumin, but not fibronectin, is selectively enhanced by chemotactic agonists and may contribute to the selective accumulation of different leucocyte subsets at the inflammatory site.     

Monocyte and Neutrophil Adhesion to Matrix Proteins is Selectively Enhanced in the Presence of Inflammatory Mediators

The authors investigated the time course of monocyte and neutrophil adhesion to fibronectin, vitronectin and albumin precoated culture wells, using mixed leucocyte populations from healthy blood donors. Moreover, the influence of chemotactic agonists on the adhesion properties as well as the quantitative expression of CD29, CD11b/CD18 and CD61 was analysed by flow cytometry. Different chemotactic agonists were used representing a classical chemotactic agonist (fMLP), and agonists with a preferential effect on monocytes (RANTES) and neutrophils (IL-8), respectively. The authors found a gradual increase in monocyte and neutrophil adhesion to all three surfaces, reaching a plateau at 15 min of incubation. Adhesion to fibronectin was significantly higher at all time points (5, 15 and 60 min, respectively) compared with vitronectin and albumin in both monocytes and neutrophils. Neutrophil adhesion to vitronectin was significantly lower at 60 min compared with 15 min. Monocyte adhesion to albumin was increased by fMLP and RANTES and to vitronectin also by IL-8. Neutrophil adhesion to albumin and vitronectin was increased by fMLP and IL-8, but not RANTES. The adhesion to fibronectin was not altered by any of the chemotactic agonists used. The quantitative levels of CD11b/CD18, but not CD29 and CD61, was increased by fMLP, but not RANTES nor IL-8. The authors conclude that the adhesion of human monocytes and neutrophils to vitronectin and albumin, but not fibronectin, is selectively enhanced by chemotactic agonists and may contribute to the selective accumulation of different leucocyte subsets at the inflammatory site.     

Neutrophil Chemotactic Activity and C5a Following Systemic Activation of Complement in Rats

Using ELISA analysis, rat C5a was stimulated in serum from rats undergoing systemic activation of complement after intravenous infusion of purified cobra venom factor (CVF). Biological (neutrophil chemotactic) activity was also assessed. Serum levels of C5a were directly proportional to the amount of CVF infused. C5a and neutrophil chemotactic activity, peaked by 5 min, then plateaued. In vitro addition of anti-C5a to serum samples of CVF-infused rats totally abolished chemotactic activity, indicating that all biological activity could be ascribed to C5a. Blood neutrophils obtained from CVF-infused animals showed a significant upregulation of CD11b, the increase being reduced (38%) in animals pretreated with anti-C5a. These findings indicate that infusion of CVF into rats produces generation of C5a, all chemotactic activity in serum being related to C5a. The in vivo generation of C5a is, at least inpart, responsible for upregulation of CD11b on blood neutrophils.     

T Cells Augment Monocyte and Neutrophil Function in Host Resistance against Oropharyngeal Candidiasis

The purpose of this study was to identify the cell populations involved in recovery from oral infections with Candida albicans. Monoclonal antibodies specific for CD4+ cells, CD8+ cells, and polymorphonuclear leukocytes were used to deplete BALB/c and CBA/CaH mice of the relevant cell populations in systemic circulation. Monocytes were inactivated with the cytotoxic chemical carrageenan. Mice were infected with 10(8) C. albicans yeast cells and monitored for 21 days. Systemic depletion of CD4+ and CD8+ T lymphocytes alone did not increase the severity of oral infection compared to that of controls. Oral colonization persisted in animals treated with head and neck irradiation and depleted of CD4+ T cells, whereas infections in animals that received head and neck irradiation alone or irradiation and anti-CD8 antibody cleared the infection in a comparable fashion. The depletion of polymorphonuclear cells and the cytotoxic inactivation of mononuclear phagocytes significantly increased the severity of oral infection in both BALB/c and CBA/CaH mice. High levels of interleukin 12 (IL-12) and gamma interferon (IFN-gamma) were produced by lymphocytes from the draining lymph nodes of recovering animals, whereas IL-6, tumor necrosis factor alpha, and IFN-gamma were detected in the oral mucosae of both na?ve and infected mice. The results indicate that recovery from oropharyngeal candidiasis in this model is dependent on CD4+-T-cell augmentation of monocyte and neutrophil functions exerted by Th1-type cytokines such as IL-12 and IFN-gamma.     

Interleukin-10 Down-Regulates Cathepsin B Expression in Fetal Rat Alveolar Type II Cells Exposed to Hyperoxia

Purpose

Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia.

Materials and Methods

Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR.

Results

LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia.

Conclusion

Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.     

Presence of HLA-G-Expressing Cells Modulates the Ability of Peripheral Blood Mononuclear Cells to Release Cytokines

PROBLEM: Human leukocyte antigen-G (HLA-G) is thought to be at play in maternal-fetal immune interplay during pregnancy. Whether the expression of HLA-G protein on the target cells altered the release of cytokines from effector mononuclear cells was questioned. METHOD OF STUDY: The amounts of cytokines released from peripheral blood mononuclear cells (PBMC) cocultured with or without HLA-G-expressing target cells were compared. RESULTS: When cocultured with HLA-G-expressing target cell lines, the amounts of interleukin-3 (IL-3) and interleukin-lβ (IL-1β) released from PBMC were increased, whereas the amounts of tumor necrosis factor-α (TNF-α) were decreased. CONCLUSIONS: Mononuclear cells, if cultured with HLA-G-expressing cells, modulate their ability to release cytokines, suggesting a role of HLA-G in triggering maternal-fetal immune interplay and thereby maintaining pregnancy.     

Ultrastructure of Highly Ordered Granules in Alveolar Type II Cells in Several Species

Alveolar Type II cells from seven mammalian species were examined for a protein in the rough endoplasmic reticulum (RER), which showed a multilayered, repeating motif. Each motif, 100 nm in width, comprised two parallel outer dense layers, a less dense central layer, and often 1–3 faint layers on either side of the latter. Outer layers showed periodicities at 3–4 densities/100 nm of width, while layers on either side of the central layer showed 5–7 densities/100 nm of width. RER membranes were ribosome‐free when parallel to these layers, but showed four ribosomes per motif at the growing ends: one ribosome at each outer dense layer, and one on either side of the less dense central layer. Granules appeared as single or as multiple motifs, stacked, curved, folded, or branching together within the same RER profile. Hexagons of around 30 nm in diameter with central densities were seen in tangential cuts of outer dense layers. Granule incidence varied: guinea pig > ferret > dog. Possible homologous structures occurred in rabbit and cat, but not in rat or mouse. Surfactant protein A (SP‐A), a C‐type lectin produced in Type II cells, forms trimers and bouquet‐like 18‐mer and can oligomerize further. Two pairs of SP‐A 18‐mers with carbohydrate recognition domains pointing inwardly and outwardly, stacked vertically as a column of four molecules, then repeated side by side in rows, approximated the size and layering patterns observed in these granules. Sequence analyses of SP‐A from these species showed phylogenetic distances consistent with the observed occurrence and frequency of patterned granules. Anat Rec, 301:1290–1302, 2018. © 2018 Wiley Periodicals, Inc.     

Immunotherapy of Rheumatoid Arthritis Targeting Inflammatory Cytokines and Autoreactive T Cells

Rheumatoid arthritis is a chronic disorder for which there is no known cure. Concentrating on specific elements of the abnormal immune response that characterizes the disease, scientists are reaching into biotechnology’s bag of tricks to develop immunotherapeutic techniques. This paper will present some advances in the immunotherapy of rheumatoid arthritis targeting inflammatory cytokines and autoreactive T cells.     

Inflammatory Cytokines and the Risk of Cardiovascular Complications in Type 2 Diabetes

This study evaluates peripheral blood T lymphocyte expression of inflammatory and proinflammatory cytokines as well as T regulatory (Treg) (FOXP3+CD25+CD4+) cells in type 2 diabetes (T2DM). Participants included 40 T2DM and 30 healthy control subjects. Twenty-four patients had no complications while 16 were afflicted with coronary heart disease (CHD). Relative to healthy subjects, all T2DM patients showed a significant increase in expression of CD4+IFN-ϒ+, CD4+TNF-α+, and CD4+IL-8+ T cells (P < 0.001) as well as CD4+IL-6+, CD4+IL-1β+, and IL-17+ T cells (P < 0.05) while the ratios of Treg/Th1(CD4+IFN-ϒ+) and Treg/Th-17(CD4+IL-17+) cells were significantly decreased (P < 0.05 and P < 0.01). T2DM patients with CHD showed a significant increase in CD4+IFN-ϒ+, CD4+TNF-α+, and CD4+IL-17+ T cells and a significant decrease in Treg/Th1 and Treg/IL-17 cells compared to T2DM patients without CHD (P < 0.05). In CHD-afflicted T2DM, HbA1c correlated positively with CD4+IFN-ϒ+ T cells (P < 0.01), HDL correlated negatively with each of CD4+IL-8+ T cells and CD4+IL-17+ T cells (P < 0.05), and LDL correlated positively with CD4+IL-1β+ T cells (P < 0.05). Conclusion. This study shows that hyperglycemia and dyslipidemia correlate with increased inflammatory cytokine expression and suggests the involvement of T cells in the development of diabetes and its complications.     

Heterogeneity of Procoagulant Activity and Cytokine Release in Subpopulations of Alveolar Macrophages and Monocytes

We have studied the expression of tissue factor (TF) and fibrinopeptide A (FPA) generation as well as the release capacity of TNF-, IL-1, and IL-6 in density-defined subpopulations of alveolar macrophages (AM) and monocytes (Mo). TF was equally expressed on all AM subpopulations and Mo, while the FPA-forming capacity was at the same level in low density AM as in Mo and was significantly (P < 0.05) higher in low density AM than in high density AM. The lipopolysaccharide (LPS)-induced release of TNF- was higher (P < 0.05) in high density AM than in low density AM and in Mo. IL-1 release was undetectable in unstimulated AM and in LPS-stimulated low density AM, while the LPS-induced IL-1 release in high density AM was low compared to the levels demonstrated in Mo. LPS-stimulated IL-6 release was not distinctively different in the AM subpopulations and Mo. The presented study showed that FPA generation and LPS-stimulated TNF- release were dependent on the density (i.e., maturity) of AM. This implies that a skewed distribution of AM subpopulations induced by disease processes may profoundly influence the inflammatory reactions, including extravascular activation of coagulation.     

Effect of Interleukin-8 and Monocyte Chemotactic Protein-1 on Adhesion of Circulating Granulocytes and Monocytes from Asthma Patients to Human Venous Endothelial Cells

The adhesive interactions between phagocytes and endothelial cells (EC) can be modulated by inflammatory cytokines and chemotactic proteins which are released during an inflammatory response. The aim of the present study was to investigate first whether the adhesive properties of granulocytes and monocytes from asthma patients for vascular endothelial cells differ from those of phagocytes from healthy individuals. Furthermore, we studied whether the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) can affect the binding of phagocytes to EC. No differences were observed in binding of phagocytes from asymptomatic or symptomatic asthma patients and from healthy individuals to non-stimulated or cytokine-stimulated EC. Incubation of granulocytes with IL-8 did not influence their adhesion to non-stimulated EC but inhibited the adhesion of granulocytes to IL-1-stimulated EC. Incubation of monocytes with MCP-1 did not affect their adhesion to non-stimulated or cytokine-stimulated EC. Our results indicate that adhesion of phagocytes to EC depends on the activation state of the endothelial cells but not on the origin of the phagocytes, since there were no differences in the adhesion of phagocytes from asthma patients and healthy individuals to non-stimulated or cytokine-stimulated EC.     

Calcitonin Gene-Related Peptide Attenuates Hyperoxia-Induced Oxidative Damage in Alveolar Epithelial Type II Cells Through Regulating Viability and Transdifferentiation

Inflammation - As a stem cell of alveolar epithelium, the physiological status of alveolar epithelium type II cells (AECII) after hyperoxia exposure is closely related to the occurrence of...     

Toll-Like Receptor 9 Signaling in Dendritic Cells Regulates Neutrophil Recruitment to Inflammatory Foci following Leishmania infantum Infection

Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL). This infection triggers dendritic cell (DC) activation through the recognition of microbial products by Toll-like receptors (TLRs). Among the TLRs, TLR9 is required for DC activation by different Leishmania species. We demonstrated that TLR9 is upregulated in vitro and in vivo during infection. We show that C57BL/6 mice deficient in TLR9 expression (TLR9^−/− mice) are more susceptible to infection and display higher parasite numbers in the spleen and liver. The increased susceptibility of TLR9^−/− mice was due to the impaired recruitment of neutrophils to the infection foci associated with reduced levels of neutrophil chemoattractants released by DCs in the target organs. Moreover, both Th1 and Th17 cells were also committed in TLR9^−/− mice. TLR9-dependent neutrophil recruitment is mediated via the MyD88 signaling pathway but is TIR domain-containing adapter-inducing interferon beta (TRIF) independent. Furthermore, L. infantum failed to activate both plasmacytoid and myeloid DCs from TLR9^−/− mice, which presented reduced surface costimulatory molecule expression and chemokine release. Interestingly, neutrophil chemotaxis was affected both in vitro and in vivo when DCs were derived from TLR9^−/− mice. Our results suggest that TLR9 plays a critical role in neutrophil recruitment during the protective response against L. infantum infection that could be associated with DC activation.     

Mechanisms of Signal Transduction from Receptors of Type I and Type II Cytokines

Cytokines play pivotal roles in regulation of immune responses. Signaling proteins involved in cytokine signal transduction pathways can be potential targets of toxins causing aberrant immune responses. Binding of cytokines to their specific receptors induces activation of signal transduction pathways. In this review, an overview of the cytokine/cytokine receptor system, signaling pathways activated by cytokine receptors, their regulation mechanisms, pathological conditions caused by aberrant cytokine signaling, and issues to be elucidated in the near future is provided.     

Alveolar Epithelial Type II Cells and Their Microenvironment in the Caveolin‐1‐Deficient Mouse

Caveolin‐1 (Cav‐1) is highly expressed in alveolar epithelial type I (AE1) and endothelial cells of the alveolar region of the lung. Interestingly, alveolar epithelial type II (AE2) cells that are progenitors of the AE1 cells do not express Cav‐1. We investigated whether genetic Cav‐1 deficiency alters the phenotype of AE2 cells and their microenvironment using stereology. Total number, mean volume, and subcellular composition of the AE2 cells were not altered in Cav‐1 ^?/? when compared with wild‐type mice. The alveolar septa were thickened and contained a significantly greater volume of extracellular matrix. Thus, AE2 cells as progenitors of AE1 cells are not critically involved in the severe pulmonary phenotype in Cav‐1‐deficient mice. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.