Dallas M. Hyde

An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.

1.1. The Challenges To understand normal lung function, the processes of growth and development, and the mechanisms and effects of diseases, we need information about the 3D structure of the lung. Quantification of organ structure is based upon 3D physical attributes of tissues, cells, organelles, alveoli, airways, and blood vessels. When structures of interest are inaccessible or too small to be seen macroscopically, we rely on physical or optical sections through a few representative samples taken from the large heterogeneous organ. The resulting 2D images confer incomplete information about the 3D structure, and may not accurately represent true 3D properties, leading to possible misinterpretation when measurements are made on 2D sections. Because structural quantification is often considered the “gold standard” in evaluating experimental intervention, disease severity, and treatment response, it is imperative that these quantitative methods are (1) accurate to allow meaningful interpretation of results, (2) efficient to yield adequate precision with reasonable effort, (3) of adequate statistical power to encompass inherent variability, and (4) adherent to uniform standards to facilitate comparisons among experimental groups and across different studies. The lung poses special challenges, some of which are outlined below and discussed in later sections: (a) Heterogeneity of lung structure requires standardized preparation methods. The inflated lung consists of mostly air; only 10 to 15% of its volume consists of tissue (cells, fibers, and matrix) and blood. In vivo lung volume and relative volumes of air, tissue, and blood fluctuate widely, while gravitational and nongravitational gradients cause spatial heterogeneity in structure and function. Failure to standardize physiological variables or minimize tissue distortion introduces uncertainties or errors into subsequent measurements, to the point of their being meaningless (1). Careful selection of fixation and preparation methods that minimize shrinkage obviates this problem (Section 3). (b) Selected microscopic sections should provide a fair sample of the whole organ. The practice of picking specific samples or sections often fails to account for regional heterogeneity, leading to biased conclusions with respect to the whole organ. Deliberately choosing sections that contain a particular compartment (e.g., profiles of alveolar type 2 epithelial cells) overestimates their abundance within the whole lung. Using a sampling scheme that covers all regions with equal probability alleviates this problem (Section 4). (c) Measurements made on microscopic sections must be related to the whole organ or an appropriate reference volume. Studies continue to appear that report only relative measurements (i.e., volume and surface densities or ratios) without knowledge of the lung volume. These ratios are dependent on lung inflation, and must be multiplied by absolute lung volume to obtain accurate total quantities of the structures of interest. Uncertainties regarding lung volume can bias data interpretation. For example, enlarged mean airspace size need not signify emphysema or alveolar hypoplasia; the finding could also be caused by overinflation. Careful measurement of the lung volume eliminates this error (Section 5). (d) Lung structures are irregular and their geometry easily altered by pathology and intervention. Measurements on 2D images that rely on assumed geometry may misrepresent the 3D structure. Examples include estimating alveolar size from cross-sectional areas of alveolar profiles, and reporting alveolar surface area by the length of alveolar profile boundary. These measures can severely misrepresent the 3D structure of interest. Airspace size is often inferred from the mean linear intercept (Lm), which in fact measures airspace volume-to-surface ratio and can be converted to diameter or volume only by assuming a shape factor. Airspace distortion, or selective distortion of alveolar ducts but not alveolar sacs, can invalidate shape assumptions (Section 6). (e) The number of lung cells cannot be estimated by counting their profiles on random histologic sections because larger cells have a greater probability of being sampled. For example, if experimental intervention causes selective cell hypertrophy, the increased probability of counting cell profiles will lead to wrong conclusions. Again, using stereologic methods that are free of geometric assumptions eliminates this error (Sections 6–7). (f) In contrast to acinar structures that exhibit nearly random orientation (isotropy) and homogeneous distribution, conducting airways and blood vessels exhibit preferred directions (anisotropy) and inhomogeneous distribution, which alter their sampling probability on random sections. Specific sampling procedures that account for their nonrandom nature should be employed to ensure unbiased representation on 2D sections (Section 8). (g) Assessment of endobronchial or lung biopsy specimens is limited by their nonrandom nature and a lack of external reference parameter. Endobronchial biopsy specimens are also anisotropic with distinct luminal and basal sides and with respect to airway generations. To minimize potential errors in quantification, specimens should be processed with their orientation randomized and analyzed with respect to an internal reference parameter (Section 9). (h) The new imaging techniques CT and MRI offer the possibility of obtaining high-fidelity images of lung structure in vivo that can be used for quantitative assessment of structural changes. Since their images are sections of the organ, stereology can ensure accurate measurements (Section 10). Definition of terms (section of text where term is defined) Accuracy (Sec. 1.2); ALP-sector (Sec. 2.1, item a); Anisotropy (Sec. 1.1, item f); Apparent diffusion coefficient (ADC) (Sec. 10.4.1); Arithmetic mean thickness of air-blood barrier (Sec. 6.7); Bias (Sec. 1.2); Buffons needle (Sec. 1.3); Cavalieri Principle/Method (Sec. 1.3); Coarse nonparenchyma (Sec. 6.2); Coarse parenchyma (Sec. 6.2); Computer-aided stereology systems (Sec. 2.2, item c); Connectivity of airway branching systems (Sec. 8.1); Delesse principle (Sec. 1.3); “Design-based” (Sec. 1.2); Dichotomous branching of airways (Sec. 8.1, Fig. 9A); Disector principle: physical, optical (Sec. 2.1, items d and e); “Do more less well” (Sec. 2.2, item c); Sec. 4.4; Efficiency (Sec. 4.4); Euler characteristic (Sec. 6.4); Fine nonparenchyma (Sec. 6.2; Figure 5); Fine parenchyma (Sec. 6.2; Equation 12); Fractal tree (Sec. 8.1); Fractionator sampling (Sec. 4.2.5; Figure 4); Global estimators (Sec. 2.1); “Gold standard” in fixation (Sec. 3.1); Harmonic mean thickness of air–blood barrier (Sec. 6.7); Horsfield ordering system (Sec. 8.1; Figure 9b); Isector (isotropic orientation) (Figure 4); Isotropic uniform random (IUR) sampling (Sec. 4.2.3); Isotropy (Sec. 1.1, item f); Local estimators (Sec. 2.1, item e); Mean chord length or mean linear intercept (Sec. 6.6); Monopodial airway branching (Sec. 8.1); Morphometry (Sec. 2.1); Multistage stratified morphometric analysis (Sec. 6.1); Multistage stratified sampling (Sec. 4.2.6); Nucleator (Sec. 2.1, item e); Number-weighted mean particle volume (Sec. 2.1, items e and f); Orientator (Sec. 4.2.3); Point-sampled intercept (Sec. 2.1, item e); Precision (Sec. 1.2); Reference space (Sec. 5); Reference lung volume (Sec. 5.1); “Reference trap” (Sec. 5); Relative deposition index (RDI) (Sec. 7.2); Relative labeling index (RLI) (Sec. 7.2); Rotator (Sec. 2.1, item e); Sampling (Sec. 2.1, Sec. 4); Sampling fraction (Sec. 6.4; Figure 4); Sampling procedures (Sec. 4.2); Sampling rules (Sec. 4.1); “Silver standards” in fixation technique (Sec. 3.1; Sec. 3.3); Stereology (Sec. 2.1); Strahler ordering system (Sec. 8.1; Figure 9b); Stratified uniform random (StUR) sampling (Sec. 4.2.2); Surface density (Sec 2.1, item b; Sec. 6.3); Systematic uniform random sampling (SURS) (Sec. 4.2.1); Test probes, test systems (Sec. 2.1, item a; Sec. 6.9; Figure 6); Uniform random sections (Sec. 4.2.1; Sec. 4.2.2; Sec. 4.2.3); Vertical sections (Sec. 4.2.4; Figure 3); Volume density (Sec. 2.1, item b; Sec. 6.2); Volume-weighted mean particle volume (Sec. 2.1, items e and f). Open in a separate window Figure 3. Vertical sections. (A) An arbitrary horizontal reference plane, such as a cutting board, is considered fixed and the vertical section is perpendicular to this horizontal plane. Airways selected by microdissection can be sampled by this vertical section scheme, by bisecting the airway longitudinally and laying it flat with the luminal surface up. In this orientation, the arrow that runs from base to apex of the epithelium indicates the direction of the vertical axis, V. (B) Bisected airway can be cut into strips of tissue

Effect of antibody to transforming growth factor beta on bleomycin induced accumulation of lung collagen in mice.

BACKGROUND--Increased production of transforming growth factor beta (TGF-beta) seems to have an important role in the pathophysiology of bleomycin induced lung fibrosis. This is attributed to the ability of TGF-beta to stimulate infiltration of inflammatory cells and promote synthesis of connective tissue, leading to collagen deposition. METHODS--The study was designed to evaluate the antifibrotic potential of TGF-beta antibody in mice treated with bleomycin, which is a model of lung fibrosis. Under methoxyflurane anaesthesia, each mouse received intratracheally either 50 microliters sterile isotonic saline or 0.125 units bleomycin in 50 microliters. Within five minutes after the instillation, mice received into the tail vein 100 microliters non-immune rabbit IgG, TGF-beta 2 antibody, or a combination of TGF-beta 2 and TGF-beta 1 antibodies at various dose regimens. Mice were killed 14 days after the instillation and their lungs processed for morphological and biochemical studies. RESULTS--Administration of 250 micrograms of TGF-beta 2 antibody after instillation of bleomycin followed by 100 micrograms on day 5 and 100 micrograms on day 9 significantly reduced the bleomycin induced increases in the accumulation of lung collagen from 445.8 (42.3) micrograms/lung to 336.7 (56.6) micrograms/lung at 14 days. Similarly, the combined treatment with 250 micrograms TGF-beta 2 antibody and 250 micrograms TGF-beta 1 antibody after bleomycin instillation followed by 100 micrograms of each antibody on day 5 also caused a significant reduction in bleomycin induced increases in lung collagen accumulation and myeloperoxidase activity at 14 days. CONCLUSIONS--These results suggest that TGF-beta has an important role in the aetiology of bleomycin induced lung fibrosis; the neutralisation of TGF-beta by systemic treatment with its antibodies offers a new mode of pharmacological intervention which may be useful in treating lung fibrosis.

Reduction of bleomycin induced lung fibrosis by transforming growth factor β soluble receptor in hamsters

BACKGROUND Transforming growth factor β (TGF-β) is a key mediator of collagen synthesis in the development of lung fibrosis. It has previously been shown that the administration of TGF-β antibody and TGF-β binding proteoglycan, decorin, reduced bleomycin (BL) induced lung fibrosis in animals. The present study was carried out to investigate whether intratracheal instillation of TGF-β soluble receptor (TR) would minimise the BL induced lung fibrosis in hamsters. METHODS The effect of a recombinant TR (TGFβRII) on the lung collagen accumulation was evaluated in a BL hamster model of pulmonary fibrosis. Animals were divided into four groups and intratracheally injected with saline or BL at 6.5 U/4 ml/kg followed by intratracheal instillation of phosphate buffered saline (PBS) or 4 nmol TR in 0.3 ml twice a week. Twenty days after the first intratracheal instillation the hamsters were killed for bronchoalveolar lavage (BAL) fluid, biochemical, and histopathological analyses. RESULTS Treatment of hamsters with TR after intratracheal instillation of BL significantly reduced BL induced lung fibrosis as shown by decreases in the lung hydroxyproline level and prolyl hydroxylase activity, although they were still significantly higher than those of the saline control. Histopathological examination showed a considerable decrease in BL induced fibrotic lesions by TR treatment. However, TR did not prevent the BL induced increases in total cells and protein in the BAL fluid. CONCLUSIONS These results suggest that TR has antifibrotic potential in vivo and may be useful in the treatment of fibrotic diseases where increased TGF-β is associated with excess collagen accumulation.

In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation

Thymic stromal lymphopoietin (TSLP) potently induces deregulation of Th2 responses, a hallmark feature of allergic inflammatory diseases such as asthma, atopic dermatitis, and allergic rhinitis. However, direct downstream in vivo mediators in the TSLP-induced atopic immune cascade have not been identified. In our current study, we have shown that OX40 ligand (OX40L) is a critical in vivo mediator of TSLP-mediated Th2 responses. Treating mice with OX40L-blocking antibodies substantially inhibited immune responses induced by TSLP in the lung and skin, including Th2 inflammatory cell infiltration, cytokine secretion, and IgE production. OX40L-blocking antibodies also inhibited antigen-driven Th2 inflammation in mouse and nonhuman primate models of asthma. This treatment resulted in both blockade of the OX40-OX40L receptor-ligand interaction and depletion of OX40L-positive cells. The use of a blocking, OX40L-specific mAb thus presents a promising strategy for the treatment of allergic diseases associated with pathologic Th2 immune responses.

Antifibrotic effect of decorin in a bleomycin hamster model of lung fibrosis

We reported previously that treatment with antibody to transforming growth factor-beta (TGF-beta) caused a marked attenuation of bleomycin (BL)-induced lung fibrosis (LF) in mice. Decorin (DC), a proteoglycan, binds TGF-beta and thereby down-regulates all of its biological activities. In the present study, we evaluated the antifibrotic potential of DC in a three-dose BL-hamster model of lung fibrosis. Hamsters were placed in the following groups: (1) saline (SA) + phosphate-buffered saline (PBS) (SA + PBS); (2) SA + DC; (3) BL + PBS; and (4) BL + DC. Under pentobarbital anesthesia, SA (4 mL/kg) or BL was instilled intratracheally in three consecutive doses (2.5, 2.0, 1.5 units/kg/4 mL) at weekly intervals. DC (1 mg/mL) or PBS was instilled intratracheally in 0.4 mL/hamster on days 3 and 5 following instillation of each dose of SA or BL. In week 4, hamsters received three doses of either DC or PBS every other day. The hamsters were killed at 30 days following the first instillation, and their lungs were appropriately processed. Lung hydroxyproline levels in SA + PBS, SA + DC, BL + PBS, and BL + DC groups were 965, 829, 1854, and 1387 microg/lung, respectively. Prolyl hydroxylase activities were 103, 289, and 193% of SA + PBS control in SA + DC, BL + PBS, and BL + DC groups, respectively. The myeloperoxidase activities in the corresponding groups were 222, 890, and 274% of control (0.525 units/lung). Intratracheal instillation of BL caused significant increases in these biochemical markers, and instillation of DC diminished these increases in the BL + DC group. DC treatment also caused a significant reduction in the infiltration of neutrophils in the bronchoalveolar lavage fluid (BALF) of hamsters in the BL + DC group. However, DC treatment had little effect on BL-induced increases in lung superoxide dismutase activity and lipid peroxidation and leakage of plasma proteins in the BALF of the BL + DC group. Hamsters in the BL + PBS group showed severe multifocal fibrosis and accumulation of mononuclear inflammatory cells and granulocytes. In contrast, hamsters in the BL + DC group showed mild multifocal septal thickening with aggregations of mononuclear inflammatory cells. Hamsters in both control groups (SA + PBS and SA + DC) showed normal lung structure. Frozen lung sections following immunohistochemical staining revealed an intense staining for EDA-fibronectin and collagen type I in the BL + PBS group as compared with all other groups. It was concluded that DC potentially offers a novel pharmacological intervention that may be useful in treating pulmonary fibrosis.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae)

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

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.

Repeated episodes of ozone inhalation amplifies the effects of allergen sensitization and inhalation on airway immune and structural development in Rhesus monkeys

Twenty-four infant rhesus monkeys (30 days old) were exposed to 11 episodes of filtered air (FA), house dust mite allergen aerosol (HDMA), ozone (O3), or HDMA + O3 (5 days each followed by 9 days of FA). Ozone was delivered for 8 h/day at 0.5 ppm. Twelve of the monkeys were sensitized to house dust mite allergen (Dermatophagoides farinae) at ages 14 and 28 days by subcutaneous inoculation (SQ) of HDMA in alum and intraperitoneal injection of heat-killed Bordetella pertussis cells. Sensitized monkeys were exposed to HDMA aerosol for 2 h/day on days 3-5 of either FA (n = 6) or O3 (n = 6) exposure. Nonsensitized monkeys were exposed to either FA (n = 6) or O3 (n = 6). During the exposure regimen, parameters of allergy (i.e., serum IgE, histamine, and eosinophilia), airways resistance, reactivity, and structural remodeling were evaluated. Eleven repeated 5-day cycles of inhaling 0.5 ppm ozone over a 6-month period had only mild effects on the airways of nonsensitized infant rhesus monkeys. Similarly, the repeated inhalation of HDMA by HDMA-sensitized infant monkeys resulted in only mild airway effects, with the exception of a marked increase in proximal airway and terminal bronchiole content of eosinophils. In contrast, the combined cyclic inhalation of ozone and HDMA by HDMA sensitized infants monkeys resulted in a marked increase in serum IgE, serum histamine, and airways eosinophilia. Furthermore, combined cyclic inhalation of ozone and HDMA resulted in even greater alterations in airway structure and content that were associated with a significant elevation in baseline airways resistance and reactivity. These results suggest that ozone can amplify the allergic and structural remodeling effects of HDMA sensitization and inhalation.

Distribution and leukocyte contacts of γδ T cells in the lung

Pulmonary γδ T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that γδ and αβ T cells are distributed differently in the normal mouse lung. The γδ T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of γδ T cells exhibit further variation in their tissue localization. γδ and αβ T cells frequently contact other leukocytes, but they favor different cell‐types. The γδ T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of γδ T cells with these sentinels likely is related to their protective role.

Lung Fibrosis is Ameliorated by Pirfenidone Fed in Diet After the Second Dose In A Three-Dose Bleomycin-Hamster Model

Interstitial lung fibrosis (ILF) is a life-threatening disease which has no known drug for prevention and cure. In the present study, we evaluated the antifibrotic potential of pirfenidone (PD) (5-methyl-1-phenyl-2-(1H)-pyridone) in a three-dose bleomycin (BL)-hamster model of lung fibrosis. Hamsters were intratracheally (IT) instilled with three consecutive doses of bleomycin sulfate (2.5 U/kg/5mL, 2.0 U/kg/5mL, 1.5 U/kg/3.75 mL) or an equivalent volume of saline at weekly intervals. Hamsters were fed a diet after the second dose of BL containing 0.5% PD and hamsters in the control groups were fed the same diet without the drug. The four groups were saline-instilled fed control diet (SCD); saline-instilled fed the same diet containing PD (SPD); BL-instilled fed control diet (BCD); and BL-instilled fed the diet containing PD (BPD). Hamsters were sacrificed at 28 days after IT instillation of last dose of saline or BL and their lungs processed for various assays. Lung hydroxyproline, an index of fibrosis, in SCD, SPD, BCD and BPD were 830, 804, 1609, 1235 micrograms/lung, respectively. Lung prolyl hydroxylase activities in the SPD, BCD and BPD groups were 103%, 313%, 157% of the control SCD group (5.99 x 10(4) dpm/lung/30 min) respectively. Malondialdehyde equivalent levels and superoxide dismutase activity in the corresponding groups were 99, 79, 240 and 145 nmoles/lung and 412, 433, 538 and 410 units/lung respectively. Lung myeloperoxidase activities in the corresponding groups were 56%, 179%, and 116% of the control group (0.44 units/lung). It is concluded that PD is a novel antifibrotic drug that has therapeutic potential in arresting the progression of an ongoing fibrotic process in the lung.