Essential Role of Osteopontin in Smoking-Related Interstitial Lung Diseases

Smoking-related interstitial lung diseases are characterized by the accumulation of macrophages and Langerhans cells, and fibrotic remodeling, which are linked to osteopontin (OPN) expression. Therefore, OPN levels were investigated in bronchoalveolar lavage (BAL) cells in 11 patients with pulmonary Langerhans cell histiocytosis (PLCH), 15 patients with desquamative interstitial pneumonitis (DIP), 10 patients with idiopathic pulmonary fibrosis, 5 patients with sarcoidosis, 13 otherwise healthy smokers, and 19 non-smoking controls. Furthermore, OPN overexpression was examined in rat lungs using adenoviral gene transfer. We found that BAL cells from patients with either PLCH or DIP spontaneously produced abundant amounts of OPN. BAL cells from healthy smokers produced 15-fold less OPN, and those cells from non-smoking healthy volunteers produced no OPN. BAL cells from patients with either idiopathic pulmonary fibrosis or sarcoidosis produced significantly less OPN, as compared with patients with PLCH. These data were confirmed by immunochemistry. Nicotine stimulation increased production of both OPN and granulocyte-macrophage colony stimulating factor by alveolar macrophages from smokers. Nicotinic acetylcholine receptor expression resembled the pattern of spontaneous OPN production and was dramatically increased in both PLCH and DIP. OPN overexpression in rat lungs induced lesions similar to PLCH with marked alveolar and interstitial accumulation of Langerhans cells. Our findings suggest a pathogenetic role of increased OPN production in both PLCH and DIP by promoting the accumulation of macrophages and Langerhans cells.Cigarette smoke is linked to a variety of lung diseases including chronic obstructive pulmonary disease, lung cancer, and interstitial lung diseases. Respiratory bronchiolar interstitial lung disease, desquamative interstitial pneumonitis (DIP), and pulmonary Langerhans cell histiocytosis (PLCH) belong to the group of smoking-related interstitial lung diseases.^1,2,3 Cigarette smoke is a complex mixture of more than 4000 compounds and is known to cause systemic and pulmonary effects.⁴ However, the underlying mechanisms as to how cigarette smoking leads to the changes observed in smoking-related interstitial lung diseases are largely unknown.^1,2,3Cigarette smoke induces inflammation, oxidative stress, and tissue injury, and has an important effect on the number, distribution, and activation state of macrophages and Langerhans cells.^5,6 There is a strong epidemiological link between PLCH and smoking. PLCH is characterized by the accumulation of activated Langerhans cells originating from the distal bronchiole walls.^1,2,3,7 The accumulations of Langerhans cells are poorly demarcated and extend to the adjacent alveoli, which often contain an abundance of pigmented macrophages. These areas show morphological changes similar to DIP.^7,8 In DIP, the predominant feature is the accumulation of alveolar macrophages, densely filling the alveolar lumen, combined with moderate fibrotic interstitial remodeling.^1,2As measured by bronchoalveolar lavage (BAL) in healthy individuals, cigarette smoking induces a 5- to 10-fold increase in alveolar macrophages in a dose-response curve.^9,10,11 It was shown that concentrations of granulocyte-macrophage colony stimulating factor (GMCSF) in patients with PLCH are increased,¹² but the mechanisms that lead to the expansion of the pulmonary macrophage pool and fibrosis in smokers are poorly understood.^1,2,3 Based on the findings of a microarray study, Woodruff et al¹³ have recently proposed that alveolar macrophages from smokers exhibit a distinctive macrophage activation state that is accompanied by increased OPN expression. Osteopontin is a glycoprotein found in the extracellular matrix of bone.¹⁴ However, multiple studies have reported cytokine properties of OPN in cell-mediated immunity.¹⁴ Further, OPN exhibits a strong chemotactic activity for macrophages, monocytes, Langerhans cells, and dendritic cells.^15,16,17In the context of these findings we speculated that OPN might be involved in the pathogenesis of smoking-related lung interstitial diseases. We found abundant OPN production by alveolar macrophages from patients with PLCH and DIP. Alveolar macrophages from both healthy smokers and patients with DIP and PLCH show up-regulated nicotine receptor expression as a sign of chronic nicotine stimulation. Further, nicotine directly induced OPN and GMCSF in alveolar macrophages. Our data provides evidence for a role of osteopontin in the pathogenesis of smoking- related interstitial lung diseases.     

Volume therapy with colloid solutions preserves intestinal microvascular perfusion in endotoxaemia

Colloid solutions have been suggested to improve microvascular perfusion due to their anti-inflammatory properties. Whether this also applies for the gut, an important immunological organ vulnerable to hypoperfusion is unknown. This study investigated intestinal microcirculation of endotoxaemic rats after volume therapy with colloid solutions such as hydroxyethyl starch (HES) and gelatin or isotonic saline (NaCl). In addition intestinal oxygenation and morphology as well as mesenteric leukocyte-endothelium interaction were quantified. Rats were anaesthetised with urethane and ketamine, mechanically ventilated, and monitored haemodynamically. Normotensive endotoxaemia was induced by a continuous intravenous infusion of Escherichia coli lipopolysaccharide (LPS, 1.5 mg kg(-1) h(-1)). After 1 h of LPS infusion either 6% HES (16 ml kg(-1)), 4% gelatin (16 ml kg(-1)) or 0.9% NaCl (64 ml kg(-1)) were infused for 1 h. Using intravital microscopy, functional capillary density (FCD) and red blood cell velocity (RBCV) were measured in the mucosa of the terminal ileum at baseline and 3 h after volume therapy. In another set of animals, mesenteric leukocyte-endothelium interaction was determined 3 h after volume therapy. In all animals intestinal lactate/pyruvate ratio and intestinal morphology were assessed. Three hours after volume therapy, FCD decreased in NaCl (808 [749/843] cm(-1); median [quartiles] P<0.05 versus baseline) but not in HES (995 [945/1036] cm(-1)) and gelatin (988 [867/1193] cm(-1)) groups. RBCV, lactate/pyruvate ratio and intestinal morphology did not differ among groups. Also mesenteric leukocyte-endothelium interaction was not significantly influenced by either treatment. In conclusion, early volume therapy with HES or gelatin, but not with NaCl, preserved gut microvascular perfusion during endotoxaemia but did not have a significant effect on tissue oxygenation nor morphological appearance in this experimental model. An anti-inflammatory effect of colloid solutions was not seen and fails to explain the changes in intestinal microcirculation.     

The emerging role of T cell Ig mucin 1 in alloimmune responses in an experimental mouse transplant model

T cell Ig mucin 1 (TIM-1) plays an important role in regulating immune responses in autoimmune and asthma models, and it is expressed on both Th1 and Th2 cells. Using an antagonistic TIM-1-specific antibody, we studied the role of TIM-1 in alloimmunity. A short course of TIM-1-specific antibody monotherapy prolonged survival of fully MHC-mismatched vascularized mouse cardiac allografts. This prolongation was associated with inhibition of alloreactive Th1 responses and preservation of Th2 responses. TIM-1-specific antibody treatment was more effective in Th1-type cytokine-deficient Stat4(-/-) recipients as compared with Th2-type cytokine-deficient Stat6(-/-) recipients. Subtherapeutic doses of rapamycin plus TIM-1-specific antibody resulted in allograft acceptance and prevented the development of chronic allograft vasculopathy. Allograft survival via this treatment was accompanied by a Th1- to Th2-type cytokine switch. Depletion of natural Tregs abrogated the graft-protecting effect of the TIM-1-specific antibody. Importantly, CD4(+)CD25(+) Tregs obtained from long-term survivors had enhanced regulatory activity as compared with naive CD4(+)CD25(+) Tregs. Consistent with this, TIM-1-specific antibody treatment both preserved Tregs and prevented the expansion of alloreactive effector Th1 cells in an alloreactive TCR transgenic adoptive transfer model. These studies define previously unknown functions of TIM-1 in regulating alloimmune responses in vivo and may provide a novel approach to promoting transplantation tolerance.     

Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis

Mycobacterium tuberculosis rapidly reduces nitrate, leading to the accumulation of nitrite. This characteristic served for the past 40 years to differentiate M. tuberculosis from other members of the Mycobacterium tuberculosis complex (MTBC), such as Mycobacterium bovis (non-BCG [referred to here as simply "M. bovis"]), Mycobacterium bovis BCG, Mycobacterium africanum, or Mycobacterium microti. Here, a narG deletion in M. tuberculosis showed that rapid nitrite accumulation of M. tuberculosis is mediated by narGHJI. Analysis of narG mutants of M. bovis and M. bovis BCG showed that, as in M. tuberculosis, nitrite accumulation was mediated by narGHJI, and no other nitrate reductase was involved. However, in contrast to M. tuberculosis, accumulation was delayed for several days. Comparison of the narGHJI promoter revealed that, at nucleotide -215 prior to the start codon of narG, M. tuberculosis carried a thymine residue, whereas the bovine mycobacteria carried a cytosine residue. Using LightCycler technology we examined 62 strains of M. tuberculosis, M. bovis, M. bovis BCG, M. microti, and M. africanum and demonstrated that this single nucleotide polymorphism was specific for M. tuberculosis. For further differentiation within the MTBC, we included, by using LightCycler technology, the previously described analysis of oxyR polymorphism, which is specific for the bovine mycobacteria, and the RD1 polymorphism, which is specific for M. bovis BCG. Based on these results, we suggest a LightCycler format for rapid and unambiguous diagnosis of M. tuberculosis, M. bovis, and M. bovis BCG.     

Clara cell protein 16 (CC16) gene polymorphism influences the degree of airway responsiveness in asthmatic children

BACKGROUND: Several studies have indicated linkage of chromosome 11q12-13 to asthma and associated traits. Among other candidate genes, the Clara cell protein 16 (CC16) gene maps to this region. CC16 is expressed in the bronchial epithelium and exhibits potent anti-inflammatory properties. A single-nucleotide polymorphism (SNP) in the CC16 gene (A38G) was previously associated with asthma. OBJECTIVE: We evaluated the role of the CC16 SNP in pediatric asthma and asthma severity in 2 German study populations. METHODS: The German Multicenter Allergy Study (MAS) cohort (n = 872, 94 asthmatic patients) and 112 allergic asthmatic children recruited in Freiburg, Germany, were included in the present study. Histamine provocations were performed at the age of 7 years in the MAS cohort to determine bronchial hyperreactivity; in the Freiburg study population a standardized exercise-induced decrease in FEV1 was evaluated. For genotyping, melting-curve analysis and restriction enzyme digestion were applied. RESULTS: No association of the CC16*38A allele with asthma could be observed in either study population. However, in asthmatic subjects (MAS cohort) PC(20)FEV(1) values were significantly lower in individuals homozygous or heterozygous for the CC16*38A allele compared with those in subjects with the CC16*38GG genotype (P <.05 and P <.03, respectively). Similarly, allergic asthmatic patients in the Freiburg cohort showed a significantly greater decrease in FEV1 after exercise when homozygous for the CC16*38A allele compared with that seen in asthmatic patients with the *38AG or *38GG genotype (P <.04 and P =.006, respectively). CONCLUSION: We conclude that the CC16*A38G SNP influences bronchial hyperreactivity and might be a genetic determinant of asthma severity in German children.     

Proteoglycans are potent modulators of the biological responses of eosinophils to chemokines

Chemokines play critical roles in governing the recruitment and activation of eosinophils at sites of allergic inflammation, particularly the asthmatic lung. However, we know little of how chemokine function is regulated post-translationally. Proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) side chains, are cell surface molecules and components of the extracellular matrix that are able to bind chemokines, whilst heparin is a GAG with therapeutic value in asthma. We examined whether soluble GAG could alter the actions of chemokines in assays of eosinophil activation. Heparin inhibited intracellular calcium flux, respiratory burst and chemotactic responses of eosinophils to CCL11, but not to the chemoattractant C5a, and inhibited binding of CCL11 to CCR3. Heparin also inhibited eosinophil stimulation by CCL11, CCL24, CCL7, CCL13 and CCL5 to differing degrees, which broadly correlated with their relative affinities for heparin. Heparan sulfate and dermatan sulfate, but not chondroitin sulfate, also inhibited the actions of CCL11 and CCL13 in assays of cellular shape change and chemotaxis. Following treatment with the sulfation inhibitor chlorate or proteoglycanases, no inhibition of CCL11-induced activity was observed using either eosinophils or a CCR3-expressing transfectant cell line. This suggests that cell surface proteoglycans are not necessary for signaling via CCR3. However, the GAG context in which chemokines are expressed is likely to represent an important level of regulation of allergic inflammation.     

Somatic alterations in the melanoma genome: A high‐resolution array‐based comparative genomic hybridization study

We performed DNA microarray‐based comparative genomic hybridization to identify somatic alterations specific to melanoma genome in 60 human cell lines from metastasized melanoma and from 44 corresponding peripheral blood mononuclear cells. Our data showed gross but nonrandom somatic changes specific to the tumor genome. Although the CDKN2A (78%) and PTEN (70%) loci were the major targets of mono‐allelic and bi‐allelic deletions, amplifications affected loci with BRAF (53%) and NRAS (12%) as well as EGFR (52%), MITF (40%), NOTCH2 (35%), CCND1 (18%), MDM2 (18%), CCNE1 (10%), and CDK4 (8%). The amplified loci carried additional genes, many of which could potentially play a role in melanoma. Distinct patterns of copy number changes showed that alterations in CDKN2A tended to be more clustered in cell lines with mutations in the BRAF and NRAS genes; the PTEN locus was targeted mainly in conjunction with BRAF mutations. Amplification of CCND1, CDK4, and other loci was significantly increased in cell lines without BRAF‐NRAS mutations and so was the loss of chromosome arms 13q and 16q. Our data suggest involvement of distinct genetic pathways that are driven either through oncogenic BRAF and NRAS mutations complemented by aberrations in the CDKN2A and PTEN genes or involve amplification of oncogenic genomic loci and loss of 13q and 16q. It also emerges that each tumor besides being affected by major and most common somatic genetic alterations also acquires additional genetic alterations that could be crucial in determining response to small molecular inhibitors that are being currently pursued. © 2010 Wiley‐Liss, Inc.     

Mutation of POC1B in a Severe Syndromic Retinal Ciliopathy

We describe a consanguineous Iraqi family with Leber congenital amaurosis (LCA), Joubert syndrome (JBTS), and polycystic kidney disease (PKD). Targeted next‐generation sequencing for excluding mutations in known LCA and JBTS genes, homozygosity mapping, and whole‐exome sequencing identified a homozygous missense variant, c.317G>C (p.Arg106Pro), in POC1B, a gene essential for ciliogenesis, basal body, and centrosome integrity. In silico modeling suggested a requirement of p.Arg106 for the formation of the third WD40 repeat and a protein interaction interface. In human and mouse retina, POC1B localized to the basal body and centriole adjacent to the connecting cilium of photoreceptors and in synapses of the outer plexiform layer. Knockdown of Poc1b in zebrafish caused cystic kidneys and retinal degeneration with shortened and reduced photoreceptor connecting cilia, compatible with the human syndromic ciliopathy. A recent study describes homozygosity for p.Arg106Pro_POC1B in a family with nonsyndromic cone‐rod dystrophy. The phenotype associated with homozygous p.Arg106Pro_POC1B may thus be highly variable, analogous to homozygous p.Leu710Ser in WDR19 causing either isolated retinitis pigmentosa or Jeune syndrome. Our study indicates that POC1B is required for retinal integrity, and we propose POC1B mutations as a probable cause for JBTS with severe PKD.