Local IL-17A Potentiates Early Neutrophil Recruitment to the Respiratory Tract during Severe RSV Infection

Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.     

IL-17, produced by lymphocytes and neutrophils,is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.     

Rapid up-regulation of CXC chemokines in the airways after Ag-specific CD4+ T cell activation

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.     

Pertussis Toxin Stimulates IL-17 Production in Response to Bordetella pertussis Infection in Mice

In a mouse model of respiratory tract infection by Bordetella pertussis, bacteria multiply in the airways over the first week and are then cleared over the next 3–4 weeks by the host immune response. Pertussis toxin (PT), a virulence factor secreted exclusively by B. pertussis, promotes bacterial growth in the airways by suppression and modulation of host immune responses. By comparison of wild type and PT-deficient strains, we examined the role of PT in modulating airway cytokine and chemokine responses affecting neutrophil recruitment during B. pertussis infection in mice. We found that, despite early inhibition of neutrophil recruitment by PT, high numbers of neutrophils were recruited to the airways by 4 days post-infection with the wild type strain, but not with the PT-deficient strain, and that this correlated with upregulation of neutrophil-attracting chemokine gene expression. In addition, there was similar upregulation of genes expressing the cytokines IL-17A (IL-17), TNF-α and IFN-γ, indicating a mixed Th1/Th17 response. Expression of IL-6, a cytokine involved in Th17 induction, was upregulated earlier than the IL-17 response. We showed that PT, rather than bacterial numbers, was important for induction of these responses. Flow cytometric analysis revealed that the IL-17-producing cells were macrophages and neutrophils as well as T cells, and were present predominantly in the airways rather than the lung tissue. Antibody neutralization of IL-17 significantly reduced chemokine gene expression and neutrophil recruitment to the airways, but only modestly increased peak bacterial loads. These data indicate that PT stimulates inflammatory responses by induction of Th1- and Th17-associated cytokines, including IL-17, during B. pertussis infection in mice, but a role for IL-17 in protection against the infection remains to be established.     

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12-15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.     

Pulmonary inflammation induced by subacute ozone is augmented in adiponectin-deficient mice: role of IL-17A

Pulmonary responses to ozone, a common air pollutant, are augmented in obese individuals. Adiponectin, an adipose-derived hormone that declines in obesity, has regulatory effects on the immune system. To determine the role of adiponectin in the pulmonary inflammation induced by extended (48-72 h) low-dose (0.3 parts per million) exposure to ozone, adiponectin-deficient (Adipo(-/-)) and wild-type mice were exposed to ozone or to room air. In wild-type mice, ozone exposure increased total bronchoalveolar lavage (BAL) adiponectin. Ozone-induced lung inflammation, including increases in BAL neutrophils, protein (an index of lung injury), IL-6, keratinocyte-derived chemokine, LPS-induced CXC chemokine, and G-CSF were augmented in Adipo(-/-) versus wild-type mice. Ozone also increased IL-17A mRNA expression to a greater extent in Adipo(-/-) versus wild-type mice. Moreover, compared with control Ab, anti-IL-17A Ab attenuated ozone-induced increases in BAL neutrophils and G-CSF in Adipo(-/-) but not in wild-type mice, suggesting that IL-17A, by promoting G-CSF release, contributed to augmented neutrophilia in Adipo(-/-) mice. Flow cytometric analysis of lung cells revealed that the number of CD45(+)/F4/80(+)/IL-17A(+) macrophages and γδ T cells expressing IL-17A increased after ozone exposure in wild-type mice and further increased in Adipo(-/-) mice. The IL-17(+) macrophages were CD11c(-) (interstitial macrophages), whereas CD11c(+) macrophages (alveolar macrophages) did not express IL-17A. Taken together, the data are consistent with the hypothesis that adiponectin protects against neutrophil recruitment induced by extended low-dose ozone exposure by inhibiting the induction and/or recruitment of IL-17A in interstitial macrophages and/or γδ T cells.     

Proinflammatory response of alveolar epithelial cells is enhanced by alveolar macrophage-produced TNF-alpha during pulmonary ischemia-reperfusion injury

Pulmonary ischemia-reperfusion (IR) injury entails acute activation of alveolar macrophages followed by neutrophil sequestration. Although proinflammatory cytokines and chemokines such as TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) from macrophages are known to modulate acute IR injury, the contribution of alveolar epithelial cells to IR injury and their intercellular interactions with other cell types such as alveolar macrophages and neutrophils remain unclear. In this study, we tested the hypothesis that following IR, alveolar macrophage-produced TNF-alpha further induces alveolar epithelial cells to produce key chemokines that could then contribute to subsequent lung injury through the recruitment of neutrophils. Cultured RAW264.7 macrophages and MLE-12 alveolar epithelial cells were subjected to acute hypoxia-reoxygenation (H/R) as an in vitro model of pulmonary IR. H/R (3 h/1 h) significantly induced KC, MCP-1, macrophage inflammatory protein-2 (MIP-2), RANTES, and IL-6 (but not TNF-alpha) by MLE-12 cells, whereas H/R induced TNF-alpha, MCP-1, RANTES, MIP-1alpha, and MIP-2 (but not KC) by RAW264.7 cells. These results were confirmed using primary murine alveolar macrophages and primary alveolar type II cells. Importantly, using macrophage and epithelial coculture methods, the specific production of TNF-alpha by H/R-exposed RAW264.7 cells significantly induced proinflammatory cytokine/chemokine expression (KC, MCP-1, MIP-2, RANTES, and IL-6) by MLE-12 cells. Collectively, these results demonstrate that alveolar type II cells, in conjunction with alveolar macrophage-produced TNF-alpha, contribute to the initiation of acute pulmonary IR injury via a proinflammatory cascade. The release of key chemokines, such as KC and MIP-2, by activated type II cells may thus significantly contribute to neutrophil sequestration during IR injury.     

Neonatal Streptococcus pneumoniae Infection May Aggravate Adulthood Allergic Airways Disease in Association with IL-17A

Epidemiologic studies have demonstrated that some bacteria colonization or infections in early-life increased the risk for subsequent asthma development. However, little is known about the mechanisms by which early-life bacterial infection increases this risk. The aim of this study was to investigate the effect of neonatal Streptococcus pneumoniae infection on the development of adulthood asthma, and to explore the possible mechanism. A non-lethal S. pneumoniae lung infection was established by intranasal inoculation of neonatal (1-week-old) female mice with D39. Mice were sensitized and challenged with ovalbumin in adulthood to induce allergic airways disease (AAD). Twenty-four hours later, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. Neonatal S. pneumoniae infection exacerbated adulthood hallmark features of AAD, with enhanced airway hyperresponsiveness and increased neutrophil recruitment into the airways, increased Th17 cells and interleukin (IL)-17A productions. Depletion of IL-17A by i.p. injection of a neutralizing monoclonal antibody reduced neutrophil recruitment into the airways, alleviated airway inflammation and decreased airway hyperresponsiveness. Furthermore, IL-17A depletion partially restored levels of inteferon-γ, but had no effect on the release of IL-5 or IL-13. Our data suggest that neonatal S. pneumoniae infection may promote the development of adulthood asthma in association with increased IL-17A production.     

Carbon monoxide inhibits IL-17-induced IL-6 production through the MAPK pathway in human pulmonary epithelial cells

Interleukin (IL)-17 is a proinflammatory cytokine that is produced by activated memory CD4 T cells, which regulates pulmonary neutrophil emigration by the induction of CXC chemokines and cytokines. IL-17 constitutes a potential target for pharmacotherapy against exaggerated neutrophil recruitment in airway diseases. As a cytoprotective and anti-inflammatory gaseous molecule, carbon monoxide (CO) may also regulate IL-17-induced inflammatory responses in pulmonary cells. Herein, we examine the production of cytokine IL-6 induced by IL-17 and the effect of CO on IL-17-induced IL-6 production in human pulmonary epithelial cell A549. We first show that IL-17 can induce A549 cells to release IL-6 and that CO can markedly inhibit IL-17-induced IL-6 production. IL-17 activated the ERK1/2 MAPK pathway but did not affect p38 and JNK MAPK pathways. CO exposure selectively attenuated IL-17-induced ERK1/ERK2 MAPK activation without significantly affecting either JNK or p38 MAPK activation. Furthermore, in the presence of U0126 and PD-98059, selective inhibitors of MEK1/2, IL-17-induced IL-6 production was significantly attenuated. We conclude that CO inhibits IL-17-stimulated inflammatory response via the ERK1/2-dependent pathway.     

Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis

Inflammation plays a critical role in lung disease progression in cystic fibrosis (CF). This inflammatory process is dominated by a neutrophil influx in the airways. To determine whether the accumulation of neutrophils in the airways of CF patients is associated with an altered function, we analyzed the capacity of neutrophils isolated from the lung compartment and the blood to release the major neutrophil pro- and anti-inflammatory cytokines IL-8 and IL-1-receptor antagonist (ra) spontaneously and in the presence of LPS. Comparison of cytokine production by blood neutrophils from CF patients and from control subjects showed significantly increased IL-8 and decreased IL-1ra release by CF neutrophils. Comparison of cytokine production by airway and blood neutrophils from CF patients also documented distinct profiles: the spontaneous release of IL-8 and IL-1ra by airway neutrophils was significantly higher than that from blood neutrophils. Culture in the presence of LPS failed to further enhance cytokine production. Analysis of the effect of dexamethasone confirmed the difference in the responsiveness of lung and blood neutrophils in CF. Used at a concentration effective in reducing IL-8 production by blood neutrophils, dexamethasone (10(-6) M) was unable to repress secretion of IL-8 by airway neutrophils. In addition, comparison of cytokine production by airway neutrophils from children with CF and children with dyskinetic cilia syndrome also documented distinct profiles of secretion. These results are consistent with a dysregulated cytokine production by lung and blood neutrophils in CF. They provide support to the hypothesis that not only the CF genotype but also the local environment may modify the functional properties of the neutrophils.     

IL-17 stimulates intraperitoneal neutrophil infiltration through the release of GRO alpha chemokine from mesothelial cells

IL-17 is a newly discovered cytokine implicated in the regulation of hemopoiesis and inflammation. Because IL-17 production is restricted to activated T lymphocytes, the effects exerted by IL-17 may help one to understand the contribution of T cells to the inflammatory response. We investigated the role of IL-17 in leukocyte recruitment into the peritoneal cavity. Leukocyte infiltration in vivo was assessed in BALB/Cj mice. Effects of IL-17 on chemokine generation in vitro were examined in human peritoneal mesothelial cells (HPMC). Administration of IL-17 i.p. resulted in a selective recruitment of neutrophils into the peritoneum and increased levels of KC chemokine (murine homologue of human growth-related oncogene alpha (GROalpha). Pretreatment with anti-KC Ab significantly reduced the IL-17-driven neutrophil accumulation. Primary cultures of HPMC expressed IL-17 receptor mRNA. Exposure of HPMC to IL-17 led to a dose- and time-dependent induction of GROalpha mRNA and protein. Combination of IL-17 together with TNF-alpha resulted in an increased stability of GROalpha mRNA and synergistic release of GROalpha protein. Anti-IL-17 Ab blocked the effects of IL-17 in vitro and in vivo. IL-17 is capable of selectively recruiting neutrophils into the peritoneal cavity via the release of neutrophil-specific chemokines from the peritoneal mesothelium.     

Role of IL-17A, IL-17F, and the IL-17 receptor in regulating growth-related oncogene-alpha and granulocyte colony-stimulating factor in bronchial epithelium: implications for airway inflammation in cystic fibrosis

IL-17R signaling is critical for pulmonary neutrophil recruitment and host defense against Gram-negative bacteria through the coordinated release of G-CSF and CXC chemokine elaboration. In this study, we show that IL-17R is localized to basal airway cells in human lung tissue, and functional IL-17R signaling occurs on the basolateral surface of human bronchial epithelial (HBE) cells. IL-17A and IL-17F were potent inducers of growth-related oncogene-alpha and G-CSF in HBE cells, and significant synergism was observed with TNF-alpha largely due to signaling via TNFRI. The activities of both IL-17A and IL-17F were blocked by a specific anti-IL-17R Ab, but only IL-17A was blocked with a soluble IL-17R, suggesting that cell membrane IL-17R is required for signaling by both IL-17A and IL-17F. Because IL-17A and IL-17F both regulate lung neutrophil recruitment, we measured these molecules as well as the proximal regulator IL-23p19 in the sputum of patients with cystic fibrosis (CF) undergoing pulmonary exacerbation. We found significantly elevated levels of these molecules in the sputum of patients with CF who were colonized with Pseudomonas aeruginosa at the time of pulmonary exacerbation, and the levels declined with therapy directed against P. aeruginosa. IL-23 and the downstream cytokines IL-17A and IL-17F are critical molecules for proinflammatory gene expression in HBE cells and are likely involved in the proinflammatory cytokine network involved with CF pathogenesis.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Regulatory effects of eotaxin on acute lung inflammatory injury

Eotaxin, which is a major mediator for eosinophil recruitment into lung, has regulatory effects on neutrophil-dependent acute inflammatory injury triggered by intrapulmonary deposition of IgG immune complexes in rats. In this model, eotaxin mRNA and protein were up-regulated during the inflammatory response, resulting in eotaxin protein expression in alveolar macrophages and in alveolar epithelial cells. Ab-induced blockade of eotaxin in vivo caused enhanced NF-kappaB activation in lung, substantial increases in bronchoalveolar lavage levels of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC), and increased MIP-2 and CINC mRNA expression in alveolar macrophages. In contrast, TNF-alpha levels were unaffected, and IL-10 levels fell. Under these experimental conditions, lung neutrophil accumulation was significantly increased, and vascular injury, as reflected by extravascular leak of (125)I-albumin, was enhanced. Conversely, when recombinant eotaxin was administered in the same inflammatory model of lung injury, bronchoalveolar lavage levels of MIP-2 were reduced, as was neutrophil accumulation and the intensity of lung injury. In vitro stimulation of rat alveolar macrophages with IgG immune complexes greatly increased expression of mRNA and protein for MIP-2, CINC, MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta. In the copresence of eotaxin, the increased levels of MIP-2 and CINC mRNAs were markedly diminished, whereas MIP-1alpha, MIP-1beta, TNF-alpha, and IL-1beta expression of mRNA and protein was not affected. These data suggest that endogenous eotaxin, which is expressed during the acute lung inflammatory response, plays a regulatory role in neutrophil recruitment into lung and the ensuing inflammatory damage.     

Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production.

Trypanosoma cruzi, which causes Chagas' disease, has been shown to cause polyclonal proliferation of lymphocytes after infection in vivo. This paper demonstrates that coculture of human PBMC with T. cruzi CL strain leads to proliferation of lymphocytes, which peaks on days 5 to 7 after infection. Approximately 15% of lymphocytes in culture undergo blast transformation. The proliferation of lymphoblasts can be measured by [3H]TdR incorporation, because the parasites incorporate little TdR. Parasites derived from autologous PBMC cultures or xenogeneic rat fibroblasts stimulate lymphocyte transformation similarly. By immunofluorescent cytometry, lymphoblasts from these cultures are 23 to 46% B cells (CD19+) and 39 to 64% T cells (CD3+), and approximately half of the T cells are CD4+ and half CD8+. A high percentage of lymphoblasts express MHC class II and IL-2R p55, suggesting both B and T lymphoblasts express these molecules. Anti-MHC class II and anti-IL-2R p55 mAb significantly inhibit the proliferative response of PBMC to T. cruzi. The mRNA for cytokines IL-1 beta, IL-2, IL-5, IL-6, IFN-gamma, and TNF-alpha are detected after T. cruzi coculture with PBMC, peaking on day 3. No IL-4 or IL-10 mRNA are detected. Large quantities of bioactive IL-1 and IL-6 are found in the supernatants of these PBMC. Monocytes, infected in the apparent absence of lymphocytes, assume activated morphology and accumulate mRNA for IL-1 beta, TNF-alpha, and IL-6. T cells require accessory cells to proliferate and produce cytokine mRNA. A trypsin-sensitive activity in lysates of T. cruzi stimulates lymphocyte proliferation. The data presented demonstrate that T. cruzi coculture with PBMC leads to lymphocyte proliferation, monocyte activation, and cytokine production.     

Human neutrophil-expressed CD28 interacts with macrophage B7 to induce phosphatidylinositol 3-kinase-dependent IFN-gamma secretion and restriction of Leishmania growth

We previously showed that CD28 is expressed on human peripheral blood neutrophils and plays an important role in CXCR-1 expression and IL-8-induced neutrophil migration. In this work we demonstrate that Leishmania major infection of macrophages results in parasite dose-dependent IL-8 secretion in vitro and in IL-8-directed neutrophil migration, as blocked by both anti-IL-8 and anti-IL-8R Abs, toward the L. major-infected macrophages. In the neutrophil-macrophage cocultures, both CTLA4-Ig, a fusion protein that blocks CD28-CD80/CD86 interaction, and a neutralizing anti-IFN-gamma Ab inhibit the anti-leishmanial function of neutrophils, suggesting that the neutrophil-macrophage interaction via CD28-CD80/CD86 plays an important role in the IFN-gamma-dependent restriction of the parasite growth. Cross-linking of neutrophil-expressed CD28 by monoclonal anti-CD28 Ab or B7.1-Ig or B7.2-Ig results in phosphatidylinositol 3-kinase association with CD28 and in wortmannin-sensitive but cyclosporin A-resistant induction and secretion of IFN-gamma. Whereas the neutrophils secrete IFN-gamma with CD28 signal alone, the T cells do not secrete the cytokine in detectable amounts with the same signal. Thus, neutrophil-expressed CD28 modulates not only the granulocyte migration but also induction and secretion of IFN-gamma at the site of infection where it migrates from the circulation.     

IL-17A and Serum Amyloid A Are Elevated in a Cigarette Smoke Cessation Model Associated with the Persistence of Pigmented Macrophages,Neutrophils and Activated NK Cells

While global success in cessation advocacy has seen smoking rates fall in many developed countries, persistent lung inflammation in ex-smokers is an increasingly important clinical problem whose mechanistic basis remains poorly understood. In this study, candidate effector mechanisms were assessed in mice exposed to cigarette smoke (CS) for 4 months following cessation from long term CS exposure. BALF neutrophils, CD4⁺ and CD8⁺ T cells and lung innate NK cells remained significantly elevated following smoking cessation. Analysis of neutrophil mobilization markers showed a transition from acute mediators (MIP-2α, KC and G-CSF) to sustained drivers of neutrophil and macrophage recruitment and activation (IL-17A and Serum Amyoid A (SAA)). Follicle-like lymphoid aggregates formed with CS exposure and persisted with cessation, where they were in close anatomical proximity to pigmented macrophages, whose number actually increased 3-fold following CS cessation. This was associated with the elastolytic protease, MMP-12 (macrophage metallo-elastase) which remained significantly elevated post-cessation. Both GM-CSF and CSF-1 were significantly increased in the CS cessation group relative to the control group. In conclusion, we show that smoking cessation mediates a transition to accumulation of pigmented macrophages, which may contribute to the expanded macrophage population observed in COPD. These macrophages together with IL-17A, SAA and innate NK cells are identified here as candidate persistence determinants and, we suggest, may represent specific targets for therapies directed towards the amelioration of chronic airway inflammation.     

Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1beta only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1alpha and pro-IL-1beta implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.