Cigarette smoke-induced pulmonary inflammation is attenuated in CD69-deficient mice

Cluster of differentiation 69 (CD69) has been identified as a lymphocyte early activation marker, and recent studies have indicated that CD69 mediates intracellular signals and plays an important role in various inflammatory diseases. Cigarette smoke (CS) is a strong proinflammatory stimulus that induces the release of proinflammatory mediators by recruiting macrophages and neutrophils into the lung tissue, and is one of the main risk factors for a number of chronic diseases. However, the potential role of CD69 in CS-induced pulmonary inflammation has not been determined. To address to this question, CD69-deficient (KO) and wild-type (WT) mice were subjected to CS-induced acute pulmonary inflammation. After the exposure with CS, the expression of CD69 in the lung of WT mice was significantly induced, it was predominantly observed in macrophages. In conjunction with this phenomenon, neutrophil and macrophage cell counts, and expression of several cytokines were significantly higher in the bronchoalveolar lavage fluid (BALF) of CS-exposed WT mice compared with air-exposed WT mice. Likewise, the CS-induced accumulation of inflammatory cells and cytokines expression were significantly lower in CD69-KO mice than in WT mice. These results suggest that CD69 on macrophages is involved in CS-induced acute pulmonary inflammation.     

Cigarette smoke-induced pulmonary inflammation and emphysema are attenuated in CCR6-deficient mice

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3alpha/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8(+) T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3alpha/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3alpha contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.     

Oophorectomy-induced bone loss is attenuated in MAGP1-deficient mice

Microfibril-associated glycoprotein-1 (MAGP1), together with the fibrillins, are constitutive components of vertebrate microfibrils. Mice deficient in MAGP1 (murine MAGP1 knockout animals (Mfap2(-/-)); MAGP1Δ) is appropriate develop progressive osteopenia and reduced whole bone strength, and have elevated numbers of osteoclasts lining the bone surface. Our previous studies suggested that the increased osteoclast population was associated with elevated levels of receptor activator of NF-κB ligand (RANKL), a positive regulator of osteoclast differentiation. To explore the relationship between RANKL expression and osteoclast differentiation in MAGP1 deficiency, oophorectomy (OVX) was used to stimulate RANKL expression in both WT and MAGP1Δ animals. Bone loss following OVX was monitored using whole body DEXA and in vivo μCT. While WT mice exhibited significant bone loss following OVX, percent bone loss was reduced in MAGP1Δ mice. Further, serum RANKL levels rose significantly in OVX WT mice, whereas, there was only a modest increase in RANKL following OVX in the mutant mice due to already high baseline levels. Elevated RANKL expression was normalized when cultured MAGP1Δ osteoblasts were treated with a neutralizing antibody targeting free TGFβ. These studies provide support for increased RANKL expression associated with MAGP1 deficiency and provide a link to altered TGF-β signaling as a possible causative signaling pathway regulating RANKL expression in MAGP1Δ osteoblasts.     

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.     

Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus Enterotoxin B in mice

Background

Cigarette smoke (CS) is a major risk factor for the development of COPD. CS exposure is associated with an increased risk of bacterial colonization and respiratory tract infection, because of suppressed antibacterial activities of the immune system and delayed clearance of microbial agents from the lungs. Colonization with Staphylococcus aureus results in release of virulent enterotoxins, with superantigen activity which causes T cell activation.

Objective

To study the effect of Staphylococcus aureus enterotoxin B (SEB) on CS-induced inflammation, in a mouse model of COPD.

Methods

C57/Bl6 mice were exposed to CS or air for 4 weeks (5 cigarettes/exposure, 4x/day, 5 days/week). Endonasal SEB (10 μg/ml) or saline was concomitantly applied starting from week 3, on alternate days. 24 h after the last CS and SEB exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and lung tissue were collected.

Results

Combined exposure to CS and SEB resulted in a raised number of lymphocytes and neutrophils in BAL, as well as increased numbers of CD8⁺ T lymphocytes and granulocytes in lung tissue, compared to sole CS or SEB exposure. Moreover, concomitant CS/SEB exposure induced both IL-13 mRNA expression in lungs and goblet cell hyperplasia in the airway wall. In addition, combined CS/SEB exposure stimulated the formation of dense, organized aggregates of B- and T- lymphocytes in lungs, as well as significant higher CXCL-13 (protein, mRNA) and CCL19 (mRNA) levels in lungs.

Conclusions

Combined CS and SEB exposure aggravates CS-induced inflammation in mice, suggesting that Staphylococcus aureus could influence the pathogenesis of COPD.     

Allergen-induced airway hyperreactivity is diminished in CD81-deficient mice

We demonstrated previously that CD81(-/-) mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81(-/-) BALB/c mice and CD81(+/+) littermates were sensitized i.p. and challenged intranasally with OVA. Although wild type developed severe AHR, CD81(-/-) mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81(-/-) T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81(-/-) T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81(-/-) mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81(-/-) and CD81(+/+) mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production.     

Role of CD69 in the pathogenesis of elastase-induced pulmonary inflammation and emphysema

Cluster of differentiation 69 (CD69), known as an early activation marker of lymphocytes, has been demonstrated to regulate inflammatory events in various disease models. Although the increased number of CD69-expressed T lymphocytes in the lungs of patients with chronic obstructive pulmonary disease (COPD) has been reported, a functional role of CD69 in the pathogenesis of COPD remains unknown. To address to this question, CD69-deficient (CD69KO) mice and wild-type (WT) mice were subjected to a mouse model of porcine pancreatic elastase (PPE)-induced pulmonary inflammation and emphysema. In the two genotypes, PPE increased counts of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) and induced emphysematous changes in the lung, whereas those two pathological signs were significantly enhanced in CD69KO mice compared to WT mice. Moreover, the PPE-induced levels of IL-17 and IL-6 in BALF were significantly higher in CD69KO mice than in WT mice at the acute inflammatory phase. Immunofluorescent studies showed that IL-17 and IL-6 were predominantly expressed in CD4⁺ and γδ T cells and macrophages, respectively. Concomitant administration of IL-17- and IL-6-neutralizing antibodies significantly attenuated the PPE-induced emphysematous changes in the two genotypes. These findings suggest that CD69 negatively regulates the development of PPE-induced emphysema in part at least through modulating function of IL-17-producing T cells.     

CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice

Increased numbers of T lymphocytes are observed in the lungs of patients with chronic obstructive pulmonary disease, but their role in the disease process is not known. We investigated the role of CD8+ T cells in inflammatory cell recruitment and lung destruction in a cigarette smoke-induced murine model of emphysema. In contrast to wild-type C57BL/6J mice that displayed macrophage, lymphocyte, and neutrophil recruitment to the lung followed by emphysema in response to cigarette smoke, CD8+ T cell-deficient (CD8-/-) mice had a blunted inflammatory response and did not develop emphysema when exposed to long-term cigarette smoke. Further studies supported a pathogenetic pathway whereby the CD8+ T cell product, IFN-gamma-inducible protein-10, induces production of macrophage elastase (matrix metalloproteinase 12) that degrades elastin, both causing lung destruction directly and generating elastin fragments that serve as monocyte chemokines augmenting macrophage-mediated lung destruction. These studies demonstrate a requirement for CD8+ T cells for the development of cigarette smoke-induced emphysema and they provide a unifying pathway whereby CD8+ T cells are a central regulator of the inflammatory network in chronic obstructive pulmonary disease.     

Cigarette smoke-induced lung emphysema in mice is associated with prolyl endopeptidase, an enzyme involved in collagen breakdown

There is increasing evidence that the neutrophil chemoattractant proline-glycine-proline (PGP), derived from the breakdown of the extracellular matrix, plays an important role in neutrophil recruitment to the lung. PGP formation is a multistep process involving neutrophils, metalloproteinases (MMPs), and prolyl endopeptidase (PE). This cascade of events is now investigated in the development of lung emphysema. A/J mice were whole body exposed to cigarette smoke for 20 wk. After 20 wk or 8 wk after smoking cessation, animals were killed, and bronchoalveolar lavage fluid and lung tissue were collected to analyze the neutrophilic airway inflammation, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels. Lung tissue degradation was assessed by measuring the mean linear intercept. Additionally, we investigated the effect of the peptide L-arginine-threonine-arginine (RTR), which binds to PGP sequences, on the smoke-induced neutrophil influx in the lung after 5 days of smoke exposure. Neutrophilic airway inflammation was induced by cigarette smoke exposure. MMP-8 and MMP-9 levels, PE activity, and PGP levels were elevated in the lungs of cigarette smoke-exposed mice. PE was highly expressed in epithelial and inflammatory cells (macrophages and neutrophils) in lung tissue of cigarette smoke-exposed mice. After smoking cessation, the neutrophil influx, the MMP-8 and MMP-9 levels, the PE activity, and the PGP levels were decreased or reduced to normal levels. Moreover, RTR inhibited the smoke-induced neutrophil influx in the lung after 5 days' smoke exposure. In the present murine model of cigarette smoke-induced lung emphysema, it is demonstrated for the first time that all relevant components (neutrophils, MMP-8, MMP-9, PE) involved in PGP formation from collagen are upregulated in the airways. Together with MMPs, PE may play an important role in the formation of PGP and thus in the pathophysiology of lung emphysema.     

Induction of autoantibodies against lung matrix proteins and smoke-induced inflammation in mice

Background

Despite recommendations for outpatient management, low risk patients with lower respiratory tract infections (LRTIs) are often hospitalized. This survey analyzed perceptions of physicians, nurses, patients and relatives about feasibility of outpatient management and required duration of hospital stay.

Methods

We performed a prospective, observational questionnaire survey in hospitalized patients with LRTI as part of a multicenter trial. Treating physicians and nurses, patients and their relatives were asked on admission and before discharge about feasibility of outpatient treatment over 5 dimensions (medical, nursing, organizational factors, and patients' and relatives' preferences) using continuous scales.

Results

On admission, 12.6% of physicians, 15.1% of nurses, 18.0% of patients and 5.2% of relatives believed that outpatient treatment would be possible. Before hospital discharge, 31.1% of physicians, 32.2% of nurses, 11.6% of patients and 4.1% of relatives thought that earlier discharge would have been feasible. Medical factors were the most frequently perceived motives for inpatient management. These perceptions were similar in all LRTI subgroups and independent of disease severity and associated expected mortality risks as assessed by the Pneumonia Severity Index (PSI).

Conclusion

Independent of type and severity of respiratory tract infection, the misperceived high severity and expected mortality and morbidity were the predominant reasons why treating physicians, nurses, patients and their relatives unanimously believed that inpatient management was necessary. Better assessment and communication about true expected medical risks might contribute to a pathway to shorten in-hospital days and to introduce a more risk-targeted and individually tailored allocation of health-care resources.

Trial Registration

NCT00350987     

Delayed onset of inflammation in protease-activated receptor-2-deficient mice

Endothelial surface expression of P-selectin and subsequent leukocyte rolling in venules can be induced by mast cell-derived histamine and binding of thrombin to protease-activated receptor-1 (PAR1). We hypothesized that activation of endothelial PAR2 by mast cell tryptase or other proteases also contributes to inflammatory responses. Leukocyte rolling flux and rolling velocity were assessed by intravital microscopy of the cremaster muscles of wild-type mice following perivenular micropipette injections of a control (LSIGRL) or PAR2-activating (SLIGRL) oligopeptide. Injection of SLIGRL increased mean rolling leukocyte flux fraction from 34 +/- 11 to 71 +/- 24% (p < 0.05) and decreased mean rolling velocity from 63 +/- 29 to 32 +/- 2 micrometer/s (p < 0.05). No significant changes occurred with control peptide injection. To further evaluate the role of PAR2 in inflammatory responses, PAR2-deficient mice were generated by gene targeting and homologous recombination. Perivenular injections of SLIGRL resulted in only a small increase in rolling leukocyte flux fraction (from 21 +/- 8 to 30 +/- 2%) and no change in rolling velocity. Leukocyte rolling after surgical trauma was assessed in 9 PAR2-deficient and 12 wild-type mice. Early (0-15 min) after surgical trauma, the mean leukocyte rolling flux fraction was lower (10 +/- 3 vs 30 +/- 6%, p < 0.05) and mean rolling velocity was higher (67 +/- 46 vs 52 +/- 36 micrometer/s, p < 0.01) in PAR2-deficient compared with control mice. The defect in leukocyte rolling in PAR2-deficient mice did not persist past 30 min following surgical trauma. These results indicate that activation of PAR2 produces microvascular inflammation by rapid induction of P-selectin-mediated leukocyte rolling. In the absence of PAR2, the onset of inflammation is delayed.     

Plasmodium yoelii sporozoites infect CD36-deficient mice

Sinnis, P. and Febbraio, M. 2001. Plasmodium yoelii sporozoites infect CD36-deficient mice. Experimental Parasitology100, 12-16.     

Dehydration anorexia is attenuated in oxytocin-deficient mice

Evidence in rats suggests that central oxytocin (OT) signaling pathways contribute to suppression of food intake during dehydration (i.e., dehydration anorexia). The present study examined water deprivation-induced dehydration anorexia in wild-type and OT -/- mice. Mice were deprived of food alone (fasted, euhydrated) or were deprived of both food and water (fasted, dehydrated) for 18 h overnight. Fasted wild-type mice consumed significantly less chow during a 60-min refeeding period when dehydrated compared with their intake when euhydrated. Conversely, fasting-induced food intake was slightly but not significantly suppressed by dehydration in OT -/- mice, evidence for attenuated dehydration anorexia. In a separate experiment, mice were deprived of water (but not food) overnight for 18 h; then they were anesthetized and perfused with fixative for immunocytochemical analysis of central Fos expression. Fos was elevated similarly in osmo- and volume-sensitive regions of the basal forebrain and hypothalamus in wild-type and OT -/- mice after water deprivation. OT-positive neurons expressed Fos in dehydrated wild-type mice, and vasopressin-positive neurons were activated to a similar extent in wild-type and OT -/- mice. Conversely, significantly fewer neurons within the hindbrain dorsal vagal complex were activated in OT -/- mice after water deprivation compared with activation in wild-type mice. These findings support the view that OT-containing projections from the hypothalamus to the hindbrain are necessary for the full expression of compensatory behavioral and physiological responses to dehydration.     

Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-gamma and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-alphabeta, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2(-/-)) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2(-/-) mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2(-/-) mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4(+) T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2(-/-) mice. Adoptive transfer experiments revealed that CD4(+) T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2(-/-) mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2(-/-) mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2(-/-) mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.     

Impairment of bleomycin-induced lung fibrosis in CD28-deficient mice

Lung fibrosis is an important pulmonary disease with a high mortality rate, but its pathophysiological mechanism has not been fully clarified. Various types of cells have been implicated in the development of lung fibrosis, including T cells. However, the contribution of functional molecules expressed on T cells to the development of lung fibrosis remains largely unknown. In this study, we determined whether costimulation via CD28 on T cells was crucial for the development of lung fibrosis by intratracheally administering bleomycin into CD28-deficient mice. Compared with wild-type mice, the CD28-deficient mice showed markedly impaired lung fibrosis after injection with low doses of bleomycin, as judged by histological changes and hydroxyproline content in the lungs. In addition, bleomycin-induced T cell infiltration into the airways and production of several cytokines and chemokines including IL-5 were also impaired in the CD28-deficient mice. Furthermore, adoptive transfer of CD28-positive T cells from wild-type mice recovered the impaired bleomycin-induced lung fibrosis in CD28-deficient mice. These findings suggest that the CD28-mediated T cell costimulation plays a critical role in the development of lung fibrosis, possibly by regulating the production of cytokines and chemokines in the lung. Thus, manipulation of the CD28-mediated costimulation could be a potential therapeutic strategy for the prevention of lung fibrosis.     

Reduced early alcohol-induced liver injury in CD14-deficient mice

Activation of Kupffer cells by gut-derived endotoxin is associated with alcohol-induced liver injury. Recently, it was shown that CD14-deficient mice are more resistant to endotoxin-induced shock than wild-type controls. Therefore, this study was designed to investigate the role of CD14 receptors in early alcohol-induced liver injury using CD14 knockout and wild-type BALB/c mice in a model of enteral ethanol delivery. Animals were given a high-fat liquid diet continuously with ethanol or isocaloric maltose-dextrin as control for 4 wk. The liver to body weight ratio in wild-type mice (5.8 +/- 0.3%) was increased significantly by ethanol (7.3 +/- 0.2%) but was not altered by ethanol in CD14-deficient mice. Ethanol elevated serum alanine aminotransferase levels nearly 3-fold in wild-type mice, but not in CD14-deficient mice. Wild-type and knockout mice given the control high-fat diet had normal liver histology, whereas ethanol caused severe liver injury (steatosis, inflammation, and necrosis; pathology score = 3.8 +/- 0.4). In contrast, CD14-deficient mice given ethanol showed minimal hepatic changes (score = 1.6 +/- 0.3, p < 0.05). Additionally, NF-kappa B, TGF-beta, and TNF-alpha were increased significantly in wild-type mice fed ethanol but not in the CD14 knockout. Thus, chronic ethanol feeding caused more severe liver injury in wild-type than CD14 knockouts, supporting the hypothesis that endotoxin acting via CD14 plays a major role in the development of early alcohol-induced liver injury.