Cigarette Smoke and DNA Cleavage Promote Lung Inflammation and Emphysema.

Smoking-related lung diseases are among the most preventable and incurable ailments in the world. Smokers are at increased risk of developing chronic obstructive pulmonary disease that can be further complicated by emphysema and lung cancer. A subset of former smokers shows persistent lung inflammation and progressive loss of lung function, indicating a role for activation of acquired immunity in smoking-induced lung diseases. In addition to the well-established noxious effects of volatile compounds in cigarette smoke, incomplete combustion of tobacco generates nano-sized carbon black (nCB) that accumulate in lung myeloid dendritic cells and macrophages. Experimentally, intra-nasal instillation nCB can cause airway inflammation and emphysema in mice, underscoring their pathogenic role in inflammatory lung diseases. High throughput analyses of macrophages that have engulfed nCB reveal de novo activation of DNA repair enzymes, and histological studies provide evidence for DNA double-stranded breaks. Emphysematous lung myeloid dendritic cells that contain nCB express pro-inflammatory cytokines, and can efficiently differentiate naive CD4 T cells to interferon-g-secreting T helper 1 and interleukin 17A expressing cell subsets. Together these findings indicate that nCB accumulation in lung innate immune cells can initiate and sustain lung inflammation and promote emphysema development.

Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10.

The mechanism by which the mammalian mother accepts the implanting fetus as an allograft remains unexplained, but is likely to be the result of a combination of factors. Mononuclear cytotrophoblasts, the specialized fetal cells of the placenta that invade the uterus, play an important role. These cells express HLA-G, an unusual major histocompatibility complex class I-B molecule, and secrete cytokines and pregnancy-specific proteins that can regulate immune function. We investigated whether cytotrophoblasts secrete interleukin 10 (IL-10), a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions. Cytotrophoblasts from all stages of pregnancy produced IL-10 in vitro, but neither placental fibroblasts nor choriocarcinoma (malignant trophoblast) cell lines did so. Spontaneous IL-10 production averaged 650, 853, and 992 pg/10(6) cells in the first, second, and third trimesters of pregnancy, respectively. IL-10 secretion dropped approximately 10-fold after the first 24 h of culture, and was paralleled by a decrease in messenger RNA. IL-10 messenger RNA was detected in biopsies of the placenta and the portion of the uterus that contains invasive cytotrophoblasts, suggesting that this cytokine is also produced in vivo. IL-10 secreted by cytotrophoblasts in vitro is bioactive, as determined by its ability to suppress interferon gamma production in an allogeneic mixed lymphocyte reaction. We conclude that human cytotrophoblast IL-10 may be an important factor that contributes to maternal tolerance of the allogeneic fetus.

Interleukin-10 is a natural suppressor of cytokine production and inflammation in a murine model of allergic bronchopulmonary aspergillosis.

We have used interleukin-10 (IL-10) gene knockout mice (IL-10-/-) to examine the role of endogenous IL-10 in allergic lung responses to Aspergillus fumigatus Ag. In vitro restimulated lung cells from sensitized IL-10-/- mice produced exaggerated amounts of IL-4, IL-5, and interferon-gamma (IFN-gamma) compared with wild-type (WT) lung cells. In vivo, the significance of IL-10 in regulating responses to repeated A. fumigatus inhalation was strikingly revealed in IL-10-/- outbred mice that had a 50-60% mortality rate, while mortality was rare in similarly treated WT mice. Furthermore, IL-10-/- outbred mice exhibited exaggerated airway inflammation and heightened levels of IL-5 and IFN-gamma in bronchoalveolar lavage (BAL) fluids. In contrast, the magnitude of the allergic lung response was similar in intranasally (i.n.) sensitized IL-10-/- and wild-type mice from a different strain (C57BL/6). Using a different route of priming (intraperitoneal) followed by one i.n. challenge we found that IL-10-/- C57BL/6 mice had heightened eosinophilic airway inflammation, BAL-IL-5 levels, and numbers of alphabetaT cells in the lung tissues compared with WT mice. We conclude that IL-10 can suppress inflammatory Th2-like lung responses as well as Th1-like responses given the constraints of genetic background and route of priming.

Beta 2-microglobulin-dependent NK1.1+ T cells are not essential for T helper cell 2 immune responses.

A number of investigations have established the critical role of interleukin 4 (IL-4) in mediating the development of T helper (Th)2 effector cells in vitro and in vivo. Despite intensive study, the origin of the IL-4 required for Th2 priming and differentiation remains unclear. Natural killer (NK)1.1+ alpha/beta T cell receptor+ T(NT) cells, a unique lineage of cells capable of producing large amounts of IL-4 after activation in vivo, are important candidates for directing Th2 priming. These cells are selected by the nonpolymorphic major histocompatibility complex (MHC) class I molecule, CD1, and are deficient in beta 2-microglobulin (beta 2m)-null mice. We used beta 2m-deficient mice on both BALB/c and C57BL/6 backgrounds to examine their capacity to mount Th2 immune responses after challenge with a number of well-characterized antigens administered by a variety of routes. As assessed by immunization with protein antigen, infection with Leishmania major, embolization with eggs of Schistosoma mansoni, intestinal infection with Nippostrongylus brasiliensis, or induction of airway hyperreactivity to aerosolized antigen, beta 2m-deficient mice developed functional type 2 immune responses that were not substantially different than those in wild-type mice. Production of IL-4 and the generation of immunoglobulin E (IgE) and eosinophil responses were preserved as assessed by a variety of assays. Collectively, these results present a comprehensive analysis of type 2 immune responses in beta 2m-deficient mice, and indicate that beta 2m-dependent NT cells are not required for Th2 development in vivo.

Interleukin 4, but not interleukin 5 or eosinophils, is required in a murine model of acute airway hyperreactivity.

Reversible airway hyperreactivity underlies the pathophysiology of asthma, yet the precise mediators of the response remain unclear. Human studies have correlated aberrant activation of T helper (Th) 2-like effector systems in the airways with disease. A murine model of airway hyperreactivity in response to acetylcholine was established using mice immunized with ovalbumin and challenged with aerosolized antigen. No airway hyperractivity occurred in severe combined immunodeficient mice. Identically immunized BALB/c mice developed an influx of cells, with a predominance of eosinophils and CD4+ T cells, into the lungs and bronchoalveolar lavage fluid at the time that substantial changes in airway pressure and resistance were quantitated. Challenged animals developed marked increases in Th2 cytokine production, eosinophil influx, and serum immunoglobulin E levels. Neutralization of interleukin (IL) 4 using monoclonal antibodies administered during the period of systemic immunization abrogated airway hyperractivity but had little effect on the influx of eosinophils. Administration of anti-IL-4 only during the period of the aerosol challenge did not affect the subsequent response to acetylcholine. Finally, administration of anti-IL-5 antibodies at levels that suppressed eosinophils to < 1% of recruited cells had no effect on the subsequent airway responses. BALB/c mice had significantly greater airway responses than C57BL/6 mice, consistent with enhanced IL-4 responses to antigen in BALB/c mice. Taken together, these data implicate IL-4 generated during the period of lymphocyte priming with antigen in establishing the cascade of responses required to generate airway hyperractivity to inhaled antigen. No role for IL-5 or eosinophils could be demonstrated.

Requirement for IL-13 independently of IL-4 in experimental asthma.

The pathogenesis of asthma reflects, in part, the activity of T cell cytokines. Murine models support participation of interleukin-4 (IL-4) and the IL-4 receptor in asthma. Selective neutralization of IL-13, a cytokine related to IL-4 that also binds to the alpha chain of the IL-4 receptor, ameliorated the asthma phenotype, including airway hyperresponsiveness, eosinophil recruitment, and mucus overproduction. Administration of either IL-13 or IL-4 conferred an asthma-like phenotype to nonimmunized T cell-deficient mice by an IL-4 receptor alpha chain-dependent pathway. This pathway may underlie the genetic associations of asthma with both the human 5q31 locus and the IL-4 receptor.

Protective role of γδ T cells in cigarette smoke and influenza infection.

Airborne pathogens commonly trigger severe respiratory failure or death in smokers with lung disease. Cigarette smoking compromises the effectiveness of innate immunity against infections but the underlying mechanisms responsible for defective acquired immune responses in smokers remains less clear. We found that mice exposed to chronic cigarette smoke recovered poorly from primary Influenza A pneumonia with reduced type I and II interferons (IFNs) and viral-specific immunoglobulins, but recruited γδ T cells to the lungs that predominantly expressed interleukin 17A (IL-17A). Il-17a-/- mice exposed to smoke and infected with Influenza A also recruited γδ T cells to the lungs, but in contrast to wild-type mice, expressed increased IFNs, made protective influenza-specific antibodies, and recovered from infection. Depletion of IL-17A with blocking antibodies significantly increased T-bet expression in γδ T cells and improved recovery from acute Influenza A infection in air, but not smoke-exposed mice. In contrast, when exposed to smoke, γδ T cell deficient mice failed to mount an effective immune response to Influenza A and showed increased mortality. Our findings demonstrate a protective role for γδ T cells in smokers and suggest that smoke-induced increase in IL-17A inhibits the transcriptional programs required for their optimal anti-viral responses. Cigarette smoke induces IL-17A expression in the lungs and inhibits γδ T-cell-mediated protective anti-viral immune responses.

IL-13 in allergy: home at last.

The immune response to allergic challenge is thought to include important effects mediated by cytokines. Of considerable interest to immunologists and parasitologists is the distinction between two closely related cytokines, IL-4 and IL-13. Although overlapping functions are inevitable, studies over the past year reveal a distinct role for IL-13 in mediating the physiologic response to a diverse array of allergens and parasites.

Activation of C3a receptor is required in cigarette smoke-mediated emphysema.

Exposure to cigarette smoke can initiate sterile inflammatory responses in the lung and activate myeloid dendritic cells (mDCs) that induce differentiation of T helper type 1 (Th1) and Th17 cells in the emphysematous lungs. Consumption of complement proteins increases in acute inflammation, but the contribution of complement protein 3 (C3) to chronic cigarette smoke-induced immune responses in the lung is not clear. Here, we show that following chronic exposure to cigarette smoke, C3-deficient (C3(-/-)) mice develop less emphysema and have fewer CD11b(+)CD11c(+) mDCs infiltrating the lungs as compared with wild-type mice. Proteolytic cleavage of C3 by neutrophil elastase releases C3a, which in turn increases the expression of its receptor (C3aR) on lung mDCs. Mice deficient in the C3aR (C3ar(-/-)) partially phenocopy the attenuated responses to chronic smoke observed in C3(-/-) mice. Consistent with a role for C3 in emphysema, C3 and its active fragments are deposited on the lung tissue of smokers with emphysema, and smoke-exposed mice. Together, these findings suggest a critical role for C3a through autocrine/paracrine induction of C3aR in the pathogenesis of cigarette smoke-induced sterile inflammation and provide new therapeutic targets for the treatment of emphysema.

Kinetic analysis of erythrocyte Na+-K+ pump and cotransport in essential hypertension.

Alterations in red blood cell (RBC) Na+-K+ pump and Na+-K+ cotransport have been described in essential hypertension. We evaluated Na+-K+ pump and cotransport in 30 hypertensive and 26 normotensive subjects subdivided by race and family history of hypertension using an improved method to examine the kinetics of Na and K effluxes. RBCs were Na-loaded by the nystatin method to five different levels of internal Na with pump determined as ouabain-sensitive Na efflux and cotransport as furosemide-sensitive Na and K efflux. Two kinetic parameters were determined for both transport systems: the apparent affinity for Na (K0.5) and the velocity of efflux at saturating internal Na concentration (Vmax). Mean intracellular Na content in fresh RBCs (mmol/L cells) was higher in black hypertensive (12.6 +/- 1.8 mmol/L cells) and normotensive subjects (10.9 +/- 1.2 mmol/L cells) than in white hypertensive (8.7 +/- 1.0 mmol/L cells) or normotensive subjects (8.5 +/- 0.8 mmol/L cells). The Vmax and K0.5 for pump were not significantly different between study groups. The Vmax for cotransport was elevated in white hypertensive compared with normotensive subjects, but the K0.5 values were similar. Black normotensive and hypertensive subjects displayed a lower Vmax and increased K0.5 for cotransport compared with the white groups. A family history of hypertension had no influence on cotransport kinetics in blacks but did predict white normotensive and hypertensive subjects with low cotransport. The reduction in intracellular Na affinity for cotransport in black subjects may explain their higher intracellular Na in fresh RBCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Constructing polycompetitor cDNAs for quantitative PCR.

Analysis of mRNA levels using reverse transcription coupled with the polymerase chain reaction provides a powerful tool for studying cytokine regulation in cellular immunology. We report a novel method for cloning competitor cDNAs that is rapid, efficient and inexpensive. By linking multiple competitor cDNAs in tandem, polycompetitor constructs can be created that allow the use of a single reagent for individual PCR assays. Assays can be performed on minute samples of cell culture or tissue and can be reliably quantitated after routine gel electrophoresis without the use of densitometry or labeled nucleotides. The utility of this technique lies in the ability to produce a relatively inexpensive customized reagent that is simple to use and that allows for sensitive determinations of gene expression in a rapid and convenient manner. This method should allow investigators in many areas of biology to easily quantitate a broad range of important regulatory molecules.

Secondary aldosteronism.

Conditions of secondary aldosteronism are common in clinical medicine, occurring in normotensive and hypertensive settings. In some conditions such as edema disorders, this represents a partially beneficial response to restore volume and Na at the expense of hypokalemia. In RVH and malignant hypertension, the aldosteronism may be beneficial, but most evidence shows a detrimental impact. In both situations, aldosterone does not compensate fully for Na degradation and facilitates K loss. In pregnancy, aldosterone's effects is more successful for volume conservation, and the action on K is almost completely overridden by other K-sparing factors. Chronic renal failure seems to best benefit from hyperaldosteronism, but the response is limited because aldosterone must act on extrarenal targets. In iatrogenic causes of secondary aldosteronism, the effects of aldosterone are mostly detrimental. The overall conclusion supports the hypothesis that aldosterone functions best in physiologic situations, but in pathophysiologic settings it does not perfectly compensate for the basic defect. This implies that in these complex conditions, successful therapy should address the disorder in aldosterone and also the other underlying pathophysiologic mechanisms.

Assessment of operative risk for patients with advanced lung disease.

Increasingly, patients with advanced lung disease are being offered operative procedures. The assessment of the perioperative risk of these patients must include not only the assessment of their lung disease, but the assessment of the patient's cardiovascular disease, their age, and their other medical problems. Knowledge of the stress of particular surgical procedures is also of importance in risk assessment, and is addressed in this article.

Construction of polycompetitors for competitive PCR.

Many different protocols are now available for competitive polymerase chain reaction (PCR) and most rely on the use of a mimic or competitor that serves as a reference for quantitation (1-4). The success (or failure) of all these protocols is critically dependent on the design, construction, and utilization of these constructs. This protocol provides detailed instructions for developing individual mimics, or competitors, for use in competitive PCR reactions. Individual competitors can be joined together in logical order in one plasmid, producing a single reagent, or polycompetitor, with multiple specificity. Although the protocol has been used successfully in producing cytokine polycompetitors, for both human and mouse (5), it should work well for almost any molecule of interest, provided sequence information is available. If a polycompetitor is to be synthesized, careful planning is especially required for a trouble-free outcome. Detailed restriction-endonuclease maps of the cloning vectors and PCR products to be cloned must be used in the design of primers and to plan appropriate strategies for incorporation of individual competitor constructs. Although many different cloning vectors may be used, in order not to be too general, this protocol provides detailed information using a commercially available vector, pGEM 11Z, and steps used in the construction of a specific polycompetitor, the human polycompetitor for T-cell cytokines, pDC10. The general principles, however, are applicable to the construction of polycom-petitors for any genes, using many different commercially available vectors.

Multistrain influenza protection induced by a nanoparticulate mucosal immunotherapeutic.

All commercial influenza vaccines elicit antibody responses that protect against seasonal infection, but this approach is limited by the need for annual vaccine reformulation that precludes efficient responses against epidemic and pandemic disease. In this study we describe a novel vaccination approach in which a nanoparticulate, liposome-based agent containing short, highly conserved influenza-derived peptides is delivered to the respiratory tract to elicit potent innate and selective T cell-based adaptive immune responses. Prepared without virus-specific peptides, mucosal immunostimulatory therapeutic (MIT) provided robust, but short-lived, protection against multiple, highly lethal strains of influenza in mice of diverse genetic backgrounds. MIT prepared with three highly conserved epitopes that elicited virus-specific memory T-cell responses but not neutralizing antibodies, termed MITpep, provided equivalent, but more durable, protection relative to MIT. Alveolar macrophages were more important than dendritic cells in determining the protective efficacy of MIT, which induced both canonical and non-canonical antiviral immune pathways. Through activation of airway mucosal innate and highly specific T-cell responses, MIT and MITpep represent novel approaches to antiviral protection that offer the possibility of universal protection against epidemic and pandemic influenza.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Link between allergic asthma and airway mucosal infection suggested by proteinase-secreting household fungi.

Active fungal proteinases are powerful allergens that induce experimental allergic lung disease strongly resembling atopic asthma, but the precise relationship between proteinases and asthma remains unknown. Here, we analyzed dust collected from the homes of asthmatic children for the presence and sources of active proteinases to further explore the relationship between active proteinases, atopy, and asthma. Active proteinases were present in all houses and many were derived from fungi, especially Aspergillus niger. Proteinase-active dust extracts were alone insufficient to initiate asthma-like disease in mice, but conidia of A. niger readily established a contained airway mucosal infection, allergic lung disease, and atopy to an innocuous bystander antigen. Proteinase produced by A. niger enhanced fungal clearance from lung and was required for robust allergic disease. Interleukin 13 (IL-13) and IL-5 were required for optimal clearance of lung fungal infection and eosinophils showed potent anti-fungal activity in vitro. Thus, asthma and atopy may both represent a protective response against contained airway infection due to ubiquitous proteinase-producing fungi.