Lei Putney

IL-17 producing γδ T cells are required for a controlled inflammatory response after bleomycin-induced lung injury

Backgroundγδ T cells play a key role in the regulation of inflammatory responses in epithelial tissue, and in adaptive immunity, as γδ T cell deficient mice have a severely impaired capacity to clear lung pathogens. γδ T cells regulate the initial inflammatory response to microbial invasion and thereby protect against tissue injury. Here we examined the response of γδ T cells to lung injury induced by bleomycin, in an effort to study the inflammatory response in the absence of any adaptive immune response to a pathogen.ResultsAfter lung injury by bleomycin, we localized the γδ T cells to the lung lesions. γδ T cells were the predominant source of IL-17 (as detected by flow cytometry and real-time PCR). Moreover, γδ T cell knockout mice showed a significant reduction in cellular infiltration into the airways, reduced expression of IL-6 in the lung, and a significant delay in epithelial repair.ConclusionMouse γδ T cells produce IL-17 in response to lung injury and are required for an organized inflammatory response and epithelial repair. The lack of γδ T cells correlates with increased inflammation and fibrosis.

Pathogenesis of Mucous Cell Metaplasia in a Murine Asthma Model

Increased mucus production in asthma is an important cause of airflow obstruction during severe exacerbations. To better understand the changes in airway epithelium that lead to increased mucus production, ovalbumin-sensitized and -challenged mice were used. The phenotype of the epithelium was dramatically altered, resulting in increased numbers of mucous cells, predominantly in the proximal airways. However, the total numbers of epithelial cells per unit area of basement membrane did not change. A 75% decrease in Clara cells and a 25% decrease in ciliated cells were completely compensated for by an increase in mucous cells. Consequently, by day 22, 70% of the total epithelial cell population in the proximal airways was mucous cells. Electron microscopy illustrated that Clara cells were undergoing metaplasia to mucous cells. Conversely, epithelial proliferation, detected with 5-chloro-2-deoxyuridine immunohistochemistry, was most marked in the distal airways. Using ethidium homodimer cell labeling to evaluate necrosis and terminal dUTP nick-end labeling immunohistochemistry to evaluate apoptosis, this proliferation was accompanied by negligible cell death. In conclusion, epithelial cell death did not appear to be the stimulus driving epithelial proliferation and the increase in mucous cell numbers was primarily a result of Clara cell metaplasia.

Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.

Background Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction.

Growth of alveoli during postnatal development in humans based on stereological estimation

Alveolarization in humans and nonhuman primates begins during prenatal development. Advances in stereological counting techniques allow accurate assessment of alveolar number; however, these techniques have not been applied to the developing human lung. Based on the recent American Thoracic Society guidelines for stereology, lungs from human autopsies, ages 2 mo to 15 yr, were fractionated and isometric uniform randomly sampled to count the number of alveoli. The number of alveoli was compared with age, weight, and height as well as growth between right and left lungs. The number of alveoli in the human lung increased exponentially during the first 2 yr of life but continued to increase albeit at a reduced rate through adolescence. Alveolar numbers also correlated with the indirect radial alveolar count technique. Growth curves for human alveolarization were compared using historical data of nonhuman primates and rats. The alveolar growth rate in nonhuman primates was nearly identical to the human growth curve. Rats were significantly different, showing a more pronounced exponential growth during the first 20 days of life. This evidence indicates that the human lung may be more plastic than originally thought, with alveolarization occurring well into adolescence. The first 20 days of life in rats implies a growth curve that may relate more to prenatal growth in humans. The data suggest that nonhuman primates are a better laboratory model for studies of human postnatal lung growth than rats.

Ozone Exposure During thE Early Postnatal Period Alters the Timing and Pattern of Alveolar Growth and Development in Nonhuman Primates

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD. Anat Rec, 2012.

In Utero and Postnatal Exposure to Environmental Tobacco Smoke (ETS) Alters Alveolar and Respiratory Bronchiole (RB) Growth and Development in Infant Monkeys

The direct effect of environmental tobacco smoke (ETS) exposure in utero on the development of the lung parenchyma is not known. We used design-based stereologic methods to evaluate in utero and postnatal ETS exposure on alveolar and respiratory bronchiole (RB) development in the rhesus macaque. Methods Timed-pregnant rhesus macaques and their offspring were exposed to filtered air or various amounts of ETS during the prenatal and postnatal period. The left cranial lobe from necropsied infants was evaluated by design-based stereological methods and general pathological review. Results Infants in the in utero and six-month ETS groups had an 18% and 17% relative decrease, respectively, in alveolar number and a 57% and 33% increase, respectively, in alveolar size compared to filtered air (FA) monkeys. Lung volume positively correlated with alveolar number in the FA and six-month ETS group and negatively correlated in the in utero ETS group. The distribution of alveolar size was much more variable in the in utero group. Overall, RB volume was significantly increased in the six-month ETS group (p < .04). Conclusions Taken together, these results indicate that in utero and postnatal ETS exposure is associated with altered parenchymal lung development.

Accelerated structural decrements in the aging female rhesus macaque lung compared with males

Aging is associated with morphometric changes in the lung that lead to decreased lung function. The nonhuman primate lung has been shown to have similar architectural, morphological, and developmental patterns to that of humans. We hypothesized that the lungs of rhesus monkeys age in a pattern similar to human lungs. Thirty-four rhesus monkeys from the California National Primate Research Center were euthanized, necropsied, and the whole lungs sampled. Stereological analysis was performed to assess the morphological changes associated with age. The number of alveoli declined significantly from age 9 to 33 yr with a greater decline in females compared with males. Lungs of females contained roughly 20% more alveoli at age 9 yr than males, but by ∼30 yr of age, females had 30% fewer alveoli than males. The volume of alveolar air also showed a significant linear decrease in females relative to age, while males did not. The number-weighted mean volume of alveoli showed a significant positive correlation with age in females but not in males. The volume of alveolar duct showed a significant positive correlation with age in females, but not in males. Structural decrements due to aging in the lung were increased in the female compared with male rhesus monkey.

Effect of rapid shallow breathing on the distribution of 18O-labeled ozone reaction product in the respiratory tract of the rat.

We examined the effect of breathing pattern on ozone reaction product content within the respiratory tract. Thirty-four anesthetized, male Wistar rats were exposed to oxygen-18 (18O)-labeled ozone at 1.0 ppm for 2 h using a dual-chamber, negative-pressure ventilation system. Frequency was set at 80 (n = 9), 120 (n = 7), 160 (n = 8), or 200 (n = 10) breaths per minute (bpm), while tidal volume (Vt) was set to provide a constant minute ventilation of 72.8 ml/min/100 g body weight. Airways sampled were from the midlevel trachea and the mainstem bronchi and parenchyma of the cranial and caudal right lobes. 18O content in each airway sample was quantified and normalized to surface area. Across frequencies, there was significantly greater (p <. 05) 18O content in the trachea and bronchi (conducting airway epithelium) compared to the parenchyma sampling sites. Tracheal 18O content decreased between 80 and 160 bpm, but then underwent an increase at 200 bpm. In comparison, 18O content gradually increased between 80 and 200 bpm at the right cranial and caudal bronchi sites. Right cranial parenchymal 18O content decreased at 200 bpm compared to 80, 120, and 160 bpm. Right caudal parenchymal 18O content was relatively constant over all breathing frequencies. We concluded that the development of rapid shallow breathing from 80 to 160 bpm results in a reduced deposition of O3 in the trachea, while only mildly affecting to ozone deposition in parenchyma supplied by short and long airway paths.

Early life exposure to allergen and ozone results in altered development in adolescent rhesus macaque lungs

In rhesus macaques, previous studies have shown that episodic exposure to allergen alone or combined with ozone inhalation during the first 6 months of life results in a condition with many of the hallmarks of asthma. This exposure regimen results in altered development of the distal airways and parenchyma (Avdalovic et al., 2012). We hypothesized that the observed alterations in the lung parenchyma would be permanent following a long-term recovery in filtered air (FA) housing. Forty-eight infant rhesus macaques (30 days old) sensitized to house dust mite (HDM) were treated with two week cycles of FA, house dust mite allergen (HDMA), ozone (O3) or HDMA/ozone (HDMA+O3) for five months. At the end of the five months, six animals from each group were necropsied. The other six animals in each group were allowed to recover in FA for 30 more months at which time they were necropsied. Design-based stereology was used to estimate volumes of lung components, number of alveoli, size of alveoli, distribution of alveolar volumes, interalveolar capillary density. After 30 months of recovery, monkeys exposed to HDMA, in either group, had significantly more alveoli than filtered air. These alveoli also had higher capillary densities as compared with FA controls. These results indicate that early life exposure to HDMA alone or HDMA+O3 alters the development process in the lung alveoli.