Bronchial Submucosa Research Articles

Role of interleukin-4 and vascular cell adhesion molecule-1 in selective eosinophil migration into the airways in allergic asthma.

Recent in vitro studies have suggested that interleukin-4 (IL-4) may be involved in the preferential migration of eosinophils into the airways in allergic asthma through its capacity to selectively increase vascular cell adhesion molecule-1 (VCAM-1) expression on vessels. To test this hypothesis, we studied the expression of VCAM-1, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) on vascular endothelium in bronchial mucosal biopsies from 20 allergic asthmatics using an immunohistochemistry technique and related the observations to IL-4 levels in bronchoalveolar lavage (BAL) fluid simultaneously obtained and to eosinophil infiltration in the bronchial mucosa. IL-4 was detectable in BAL fluid from nine subjects (range, 15.1 to 110 pg/ml in 20-fold concentrated BAL fluid) (IL-4-positive asthmatics) but unmeasurable in the remaining 11 subjects (IL-4-negative asthmatics). The IL-4-positive asthmatics showed a significantly increased expression of VCAM-1 but not E-selectin and ICAM-1 on vessels as compared with both IL-4-negative asthmatics (P < 0.001) and diseased control subjects (P < 0.001). In asthmatics, VCAM-1 expression was positively correlated with BAL IL-4 levels (rs = 0.89; P < 0.0001). Moreover, there was a significant correlation between the endothelial expression of VCAM-1 and the number of eosinophils, but not neutrophils, in the bronchial submucosa (r2 = 0.76; P < 0.001). A significant correlation was also found between BAL IL-4 levels and the number of eosinophils. These results suggest that IL-4 is a VCAM-1-selective activator also in human airways and the VCAM-1-dependent pathways play a role in selective migration of eosinophils into the airways in allergic asthma, and support the hypothesis described above.

Enhanced production and gene expression of IL-5 in bronchial asthma. Possible management of atopic diseases with IL-5 specific gene transcription inhibitor.

Infiltration of various inflammatory cells into the bronchial mucosa and submucosa is a prominent pathological feature of bronchial asthma.1–3 Persistent mucosal inflammation, particularly epithelial damage caused by eosinophil-derived products, is believed to contribute to the pathogenesis of bronchial hypersensitivity.4–7 Inhalation of a relevant allergen results in an early asthmatic reaction (EAR) that subsides within 1 to 2 hours. In 40–60% of patients, this early reaction is followed after 6 to 10 hours by a late asthmatic reaction (LAR), which usually subsides during the next 1 to 2 days.8 Accumulating evidence suggests that LAR is a consequence of eosinophilic inflammation in the lung induced by a T cell cytokine, interleukin 5 (IL-5).9–15

Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness.

A murine model for antigen-induced bronchial hyperreactivity (BHR) and airway eosinophilia, two hallmarks of asthma, was developed using ovalbumin-immunized mice, which produce large amounts of IgE (named BP2, "Bons Producteurs 2," for High Line of Selection 2). A single intranasal ovalbumin challenge failed to modify the bronchial responses, despite the intense eosinophil recruitment into the bronchoalveolar lavage fluid and airways. When mice were challenged twice a day for 2 days or once a day for 10 days, BHR in response to i.v. 5-hydroxytryptamine or to inhaled methacholine was induced in BP2 mice but not in BALB/c mice. Histological examination showed that eosinophils reached the respiratory epithelium after multiple ovalbumin challenges in BP2 mice but remained in the bronchial submucosa in BALB/c mice. Total IgE titers in serum were augmented significantly with immunization in both strains, but much more so in BP2 mice. Interleukin 5 (IL-5) titers in serum and bronchoalveolar lavage fluid of BP2 mice were augmented by the antigenic provocation, and a specific anti-IL5 neutralizing antibody suppressed altogether airway eosinophilia and BHR, indicating a participation of IL-5 in its development. Our results indicate that the recruitment of eosinophils to the airways alone does not induce BHR in mice and that the selective effect on BP2 mice is related to their increased IgE titers associated with antigen-driven eosinophil migration to the epithelium, following formation and secretion of IL-5.

Comparison of leukocyte counts in sputum, bronchial biopsies, and bronchoalveolar lavage.

To determine the relationship between inflammatory cells in sputum, bronchoalveolar lavage (BAL), and bronchial mucosa, we counted the number of leukocytes in sputum, BAL, and bronchial biopsies obtained from subjects with asthma and with chronic bronchitis in stable condition or during exacerbations. Sputum was induced by inhalation of hypertonic saline in the asthma group. Spontaneous sputum was collected in the chronic bronchitis groups. Differential counts of leukocytes were performed on cytospin preparations of sputum and BAL. Eosinophils, macrophages, neutrophils, and lymphocytes were quantified in the submucosa of the bronchial biopsies. In asthma and in stable chronic bronchitis, the percentages of neutrophils were significantly higher in sputum than in BAL, whereas the opposite was true of the percentages of macrophages and lymphocytes. The lymphocyte was the predominant cell infiltrating the bronchial submucosa in all groups. BAL eosinophils correlated with submucosal and sputum eosinophils in the asthma and exacerbated chronic bronchitis groups. A similar trend was observed between submucosal and sputum eosinophils. In conclusion, the relative proportion of inflammatory cells was different in sputum, BAL, and bronchial mucosa. However, there was a fairly good agreement between the number of eosinophils counted with the three techniques in asthmatics and in exacerbated chronic bronchitics, suggesting that sputum cell analysis may be used for a noninvasive assessment of airway eosinophilia.

15-lipoxygenase immunoreactivity in normal and in asthmatic airways.

Products of the 15-lipoxygenase (15-LO) pathway of arachidonic acid metabolism such as the mono- and di-hydroxyeicosatetraenoic acids (HETEs) may contribute to the pathophysiology of allergic airway inflammation through the recruitment and activation of inflammatory cells and stimulation of glandular secretion. In this study we have examined the expression of 15-LO and its cellular localization in the asthmatic and normal bronchial mucosa. Bronchial mucosal biopsies were obtained by fiberoptic bronchoscopy from 10 patients with symptomatic allergic asthma and six normal control subjects and processed into glycolmethacrylate resin. Sections 2 microns thick were immunostained using a specific rabbit polyclonal antihuman 15-LO antibody. Strong immunoreactivity for 15-LO was present throughout the epithelium in both the asthmatic and the normal subjects, with no difference between the two groups. Cells expressing 15-LO immunoreactivity were also present in the submucosa of both groups, with a significantly greater number present in the asthmatic group (median, 15.3 cells/mm2) than in the normal group (median, 6.9 cells/mm2) (p = 0.01). The majority (85%) of the submucosal 15-LO+ cells were eosinophils. Patchy 15-LO immunoreactivity was also seen in the vascular endothelium in both groups. These findings demonstrated increased 15-LO expression in the bronchial submucosa of asthmatic subjects, and they suggest that 15-LO products in asthma originate from both bronchial epithelium and infiltrating eosinophils.

Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF-alpha-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion.

Asthma is a disease of airway inflammation and hyper-reactivity associated with lymphocytic infiltration in the bronchial submucosa. We recently demonstrated that human airway smooth muscle (ASM) cells express the cell adhesion molecules ICAM-1 and VCAM-1, which are up-regulated by cytokines such as TNF-alpha, and which mediate binding of activated T lymphocytes. In this study, we examined whether an increase in [cAMP]i, presumably via activation of cAMP-dependent protein kinase, modulates TNF-alpha-induced ICAM-1 and VCAM-1 on ASM. We found that treatment of ASM with either forskolin, which directly activates adenylyl cyclase, or with cholera toxin, which activates the heterotrimeric GTP-binding protein, Gs, inhibited TNF-alpha-induced cell adhesion molecule expression. In addition, treatment with either isoproterenol or prostaglandin E2, which activates receptors coupled to Gs and increases [cAMP]i, also inhibited TNF-alpha-induced expression of ICAM-1 and VCAM-1 on ASM. Furthermore, adhesion of activated T cells to TNF-alpha-stimulated ASM was inhibited by treating the ASM cells with either forskolin or PGE2. These data suggest that cAMP-dependent protein kinase activation decreases cytokine-induced expression of cell adhesion molecules on ASM cells, modulates T cell binding to airway myocytes and, thus, suggests novel therapeutic approaches to airway inflammation.

Bronchoscopic findings of extramural lung cancer invading the subepithelium or submucosa

Bronchoscopic findings from the main to segmental bronchi were compared with the histopathological findings in 185 resected cases of lung cancer, in order to determine which bronchoscopic features are associated with lung cancer invading the subepithelium or submucosa from beyond the bronchial wall. Carcinoma invaded the subepithelium or submucosa from beyond the bronchial wall in 43 cases (22.9%) out of the total of 185 cases. Bronchoscopic findings were evaluated in these 43 cases, and were summarized as follows: (1) The bronchoscopic findings in cases of subepithelial invasion consisted of vascular engorgement, bleeding, subepithelial tumor, and emphasized longitudinal relief; (2) irregularity of the mucosa was observed in cases of epithelial or muscular invasion; (3) indistinct bronchial cartilage was observed in cases of invasion proximal to the extramuscular layer; (4) accentuated irregular folds were observed in cases of invasion of the extramuscular or cartilage layers; and (5) edema and redness were not specific for malignancy. In addition, the occurrence of mediastinal lymph node metastasis was higher in cases of invasion to main or lobar bronchi. This result indicates that recognition of invasion of the subepithelium or submucosa of the central bronchus may be helpful in indicating the probability of mediastinal lymph node metastasis. Accordingly, there may be specific bronchoscopic findings which correlate with invasion of the bronchial subepithelium or submucosa. Accurate recognition of these findings may be useful in determining appropriate biopsy sites and may provide more information concerning selection of therapeutic strategy.

In situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of VCAM-1/VLA-4 interaction in selective eosinophil infiltration.

Eosinophils play a critical role in the pathogenesis of bronchial asthma by releasing various mediators. To understand the mechanisms of eosinophil migration to the site of inflammation, we examined the expression of adhesion molecules in the bronchial tissues of asthmatic subjects with air flow limitation. By immunohistochemical analysis, Mac-1, LFA-1, and VLA-4 were strongly positive in eosinophils and mononuclear cells infiltrated in the bronchial mucosa and submucosa. Their number was significantly increased compared with those in control tissue. Immunolocalization for ICAM-1, the ligand of Mac-1 and LFA-1, was detected in the endothelial cells of capillaries and venules, in the mononuclear cells in submucosa, and in the basal layer of the epithelium. Endothelial cells in capillaries and venules were also strongly positive for VCAM-1, the ligand of VLA-4. Immunolocalization for E-selectin was detected in some endothelial cells in capillaries and venules in bronchial submucosa, whereas there were very few positive cells in the bronchial tissues from control subjects. In situ hybridization demonstrated ICAM-1 mRNA expression in the endothelial cells and mononuclear cells in bronchial submucosa. Immunoelectron microscopy for ICAM-1, VCAM-1, and E-selectin demonstrated de novo synthesis of these molecules and their expression along the luminal cell membrane of endothelial cells. These results suggested that ICAM-1, VCAM-1, and E-selectin were newly synthesized prior to spontaneous asthma attacks, and that their expression, particularly that of VCAM-1, may play a key role in eosinophil infiltration into the airway.

Expression of endothelial cell adhesion molecules in human bronchial xenografts.

The role of endothelial cell adhesion molecules (CAMs) in the selective recruitment of leukocyte subsets to the airway remains unclear. The goal of the present study was to examine the expression of human endothelial CAM in a cytokine-induced airway inflammatory response. To accomplish this, an in vivo model of human bronchus was developed by heterotopically transplanting intact sections of human airway into severe combined immunodeficient (SCID) mice. Three weeks after transplantation, the xenografts closely resembled normal bronchus with little evidence of rejection. Less than 15% of the submucosal vessels expressed E-selectin and vascular cell adhesion molecule-1 (VCAM-1), whereas intercellular adhesion molecule-1 (ICAM-1) was present constitutively on approximately 35% of bronchial microvessels. Intrabronchial instillation of tumor necrosis factor-alpha (TNF-alpha) significantly increased expression of microvascular E-selectin to 40%, ICAM-1 to 65%, and VCAM-1 to 41%, and was accompanied by an influx of murine leukocytes into the bronchial submucosa. These results demonstrate that the human bronchial microvasculature expresses cytokine-inducible adhesion molecules. Mast cells and other resident or migratory cells that secrete TNF-alpha may thus activate the bronchial microvasculature and thereby recruit leukocytes to the airway.

T lymphocytes adhere to airway smooth muscle cells via integrins and CD44 and induce smooth muscle cell DNA synthesis.

Asthma is a disease of airway inflammation and hyperreactivity that is associated with a lymphocytic infiltrate in the bronchial submucosa. The interactions between infiltrating T lymphocytes with cellular and extracellular matrix components of the airway and the consequences of these interactions have not been defined. We demonstrate the constitutive expression of CD44 on human airway smooth muscle (ASM) cells in culture as well as in human bronchial tissue transplanted into severe combined immunodeficient mice. In contrast, basal levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression are minimal but are induced on ASM by inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha). Activated, but not resting T cells, adhere to cultured ASM; stimulation of the ASM with TNF-alpha enhanced this adhesion. Adhesion was partially blocked by monoclonal antibodies (mAb) specific for lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) on T cells and ICAM-1 and VCAM-1 on ASM cells. The observed integrin-independent adhesion was mediated by CD44/hyaluronate interactions as it was inhibited by anti-CD44 mAb 5F12 and by hyaluronidase. Furthermore, the adhesion of activated T lymphocytes induced DNA synthesis in growth-arrested ASM cells. Thus, the interaction between T cells and ASM may provide insight into the mechanisms that induce bronchial inflammation and possibly ASM cell hyperplasia seen in asthma.

Pneumonectomy for unremitting hemoptysis in unilateral absence of pulmonary artery

Unilateral absence of pulmonary artery is a rare malformation that can present as an isolated lesion or may be associated with other congenital heart defects. Clinical presentation is subtle when the lesion occurs alone, and may include hemoptysis, which results from rupture of abundant bronchial submucosal vessels perfused by enlarged systemic collaterals that supply the affected lung. Pneumonectomy is recommended as definitive treatment in such an adult patient.

Acute severe asthma: Part 1. Pathophysiology and clinical assessment.

Asthma manifests itself in a two-phase reaction. An early phase, causing bronchial muscle contraction, occurs within minutes after an antigenic stimulus. Within 6 to 18 hours, a late asthmatic response begins. This response is associated with cellular infiltration of the bronchial submucosa by eosinophils, neutrophils, and lymphocytes. The late asthmatic response is most effectively reversed by the use of corticosteroids. Predicting patient outcome solely on the basis of either physical findings, or pulmonary function tests, or historical events is frequently inadequate. Historical events, clinical assessments, and laboratory and pulmonary function measurements are best used in a complementary and integrated fashion to determine appropriate disposition of the acutely asthmatic patient.

Is there a role for cyclosporine in asthma?

Airway inflammation is a characteristic feature of asthma. After contact with a specific allergen on the surface of bronchial airway mast cells, IgE-dependent degranulation induces the early phase bronchial response, consisting of smooth muscle contraction and increased vascular permeability. The inflammatory LPR that begins 4 to 8 hours after the early phase is characterized by inflammatory cellular infiltrates in the bronchial mucosa and submucosa. The cells present in the bronchial airway wall and lumen, including mast cells, basophils, eosinophils, neutrophils, platelets, Tand B-lymphocytes, macrophages, and epithelial cells, are all involved in the pathogenesis of the inflammation in asthma. Although none of the cells can be identified as the primary effector cell, lymphocytes appear to have a critical role because of their immunoregulatory functions. Systemic glucocorticoids remain the cornerstone of the treatment of severe asthma in patients refractory to bronchodilator therapy. Their long-term use systemically, however, is associated with substantial and often irreversible adverse side effects. The favorable experience with nonsteroidal anti-inflammatory therapy of dermatologic and rheumatologic diseases has prompted studies of drugs in the treatment of severe asthma. Anti-inflammatory agents studied for their steroid-sparing capability include methotrexate, gold, nedocromil , colchicine, dapsone, hydroxychloroquine, and azathioprine, among other drugs. Only methotrexate, gold, and nedocromil have proven to

Effect of Inhaled Platelet-Activating Factor on Bronchial Inflammation in Atopic Non-Asthmatic Subjects

We have investigated the effect of inhaled platelet-activating factor (PAF) on bronchial mucosal inflammation in 6 atopic non-asthmatic subjects in a double-blind, placebo-controlled, randomised and crossover study. On 2 study periods at least 4 weeks apart, fiberoptic bronchoscopy was performed 24 h after inhalation of either 200 micrograms PAF or methacholine (control) to obtain endobronchial biopsies. Immunocytochemistry using antibodies for trypase (AA1) and eosinophil cationic protein (EG2) was performed to enumerate mast cells and eosinophils, respectively, in the bronchial submucosa. Median values of AA1+ cells and EG2+ cells did not differ significantly after inhalation of PAF or control (23.8 vs. 39 and 6 vs. 8/mm2, respectively, PAF vs. control, non-significant). Our findings suggest that within 24 h of inhaling a bronchoconstrictor dose of PAF, this agonist does not induce bronchial hyperresponsiveness or mucosal inflammation in atopic non-asthmatic subjects. However, because of the small number of subjects studied, these preliminary data should be interpreted with caution.

Immunohistochemical Localization of Peptide-containing Nerves in Human Airways: Age-related Changes

Bronchial reactivity changes during childhood, indicating possible changes in neural control. Nerves supplying the intrapulmonary airways were therefore studied in autopsy tissue from 14 normal infants (0 to 3.5 yr), 3 children (8.3 to 10.75 yr), and 4 adults (17 to 24 yr). An indirect immunofluorescence technique was used to study the distribution and relative number of nerve fibers containing the general neuronal markers protein gene product 9.5 and synaptophysin. Nerve subpopulations were identified using antisera to neuropeptide tyrosine, vasoactive intestine polypeptide, somatostatin, substance P, calcitonin gene-related peptide, and the enzyme tyrosine hydroxylase. Between birth and 3 yr, the distribution and relative number of immunoreactive nerves shown by both the general neuronal markers and specific antisera did not change. Neuropeptide tyrosine-immunoreactive nerves were the most common peptide-containing nerve subpopulation identified in the human lung, supplying bronchial smooth muscle, submucosal glands, cartilage, and submucosa. Other peptide-containing nerves exhibited distinct distribution patterns. Two differences in the airway innervation were identified between cases aged 0 to 3.5 yr and the older age groups. Relatively fewer peptide-containing nerves occurred in the adult bronchioli and respiratory unit, but the relative number of vasoactive intestinal polypeptide-containing nerves supplying the bronchial and bronchiolar smooth muscle was greater in the two older age groups. Given these apparent age-related differences in the number of peptide-containing nerves supplying the human airway, studies on the development of peptide receptors are indicated.

Effect of omental, intercostal, and internal mammary artery pedicle wraps on bronchial healing

Bronchial transection and devascularization is necessary in the course of sleeve resection or lung transplantation, leaving distal bronchial segments ischemic and subject to stricture or dehiscence. Thirty mongrel dogs underwent left lung autotransplantation. The bronnchial anastomosis was wrapped with omentum (n = 9), intercostal muscle pedicle (n = 9), or internal mammary artery pedicle grafts (n = 6). Six control animals underwent bronchial anastomosis without an external wrep. Bronchial revascurlarization by capillary ingrowth from the pedicle to the bronchial submucosal plexus was demonstrated with all three types of vascular pedicle grafts; however, more consistent and confluent vascular ingrowth was provided by internal mammary artery pedicle grafts. Additionally, the bronchial anastomotic cross-sectional area was significantly better in the internal mammary artery group (84.5 ± 3.3) as compared with that of the omental (68.4 ± 8.3), intercostal muscle (66.9 ± 10.9), or contral groups (70.2 ± 7.6). An internal mammary artery pedicle graft and the presence of dense confluent submucosal vascular ingrowth from any pedicle graft were independently predictive ( p < 0.05) of minimizing bronchial anastomotic narrowing. These data are consistent with previous findings suggesting that omental and intercostal muscle pedicle grafts promote early bronchiai revascuiarization; moreover, the data demonstrate the superiority of an internal mammary artery pedicle graft to provide submucosal vascular ingrowth and to minimize anastomotic stenosis.

Potential role of dendritic cells in bronchiolitis obliterans in heart-lung transplantation

Dendritic cells are essential for the activation of the type IV immunological reactions that are intrinsic to rejection of transplanted organs. We evaluated the number of dendritic cells in the recipient and donor portions of the trachea and donor bronchi of 6 heart-lung transplant recipients, 3 of whom had evidence of bronchiolitis obliterans, a presumed manifestation of chronic rejection of the lung. As compared with recipients without bronchiolitis obliterans, patients with it showed a significant increase in the number of S100-protein-positive dendritic cells in the tracheal and bronchial epithelium and submucosa of the donor organs. This finding of increased accessary cells in the transplanted mucosa provides support for the belief that bronchiolitis obliterans is an immunologically mediated form of chronic rejection.

Ultrastructural analysis of leukocytes recruited into the guinea pig lung by LTB4 aerosol

Two groups of naive guinea pigs were exposed to different LTB4 aerosol solutions: LTB4/saline and LTB4/BSA. Control animals were exposed to saline alone and saline/BSA. The LTB4/BSA aerosol was the most effective in recruiting leukocytes, with the maximal numbers of cells in the lung tissue at 6 hrs. Light microscopy/TEM revealed marked differences in eosinophil morphology in saline/BSA and LTB4/BSA aerosol treated animals. Occasional eosinophils in the submucosa of controls, had few pseudopodia, few cytoplasmic vesicles, and intact granules. In LTB4 treated animals, there were numerous eosinophils in the bronchial mucosa and submucosa with numerous pseudopodia, numerous cytoplasmic vesicles, and light crystalline cores.

PAF-Acether and Experimental Anaphylaxis as a Model for Asthma

Several lines of evidence indicate that platelet-activating factor (PAF-acether) is implicated in hypersensitivity reactions. Indeed, PAF-acether reproduces the features of asthma in vivo and in vitro, since it induces bronchoconstriction, hypotension, and hemoconcentration and activates platelets and leukocytes. Both PAF-acether and antigen evoke eosinophil margination and diapedesis in guinea pig lung parenchyma and bronchial submucosa. Furthermore, intradermal administration of PAF-acether to atopics induces a more intense eosinophil degranulation as compared to normal subjects. PAF-acether also induces bronchopulmonary hyperresponsiveness in various animal models and in humans. We showed that lungs from actively sensitized guinea pigs exhibit an in vitro bronchopulmonary hyperresponsiveness to PAF-acether as compared to nonsensitized animals. This phenomenon is probably due to a lung invasion by inflammatory cells or to a variation of the reactivity of resident lung cells such as alveolar macrophages. In these cells, the cyclic adenosine monophosphate content is much less increased by prostaglandin E2 and salbutamol when they are obtained from actively sensitized animals.

Diagnosis of lung allograft rejection by bronchial intraepithelial Leu-7 positive T lymphocytes

Currently there is no reliable technique for the diagnosis of lung allograft rejection. The presence of intraepithelial lymphocytes expressing the Leu-7 antigen is a specific marker of renal rejection. We examined whether immunoperoxidase techniques that detect Leu-7 positive lymphocytes could be used to diagnose lung rejection in heart-lung transplant recipients. In lungs from two autopsied patients with lung allograft rejection, numerous Leu-7 positive lymphocytes were present in the donor bronchial mucosa (32 and 65 cells/section), submucosa (23 and 80 cells/section), and submucosal glands (7 and 19 cells/section). These Leu-7 positive lymphocytes were associated with proximal airway injury, including squamous metaplasia, destruction of submucosal glands, and ulceration. In one case, there was bronchiectasis. Both cases also had distal airway bronchiolitis obliterans. In contrast, Leu-7 positive lymphocytes were not identified in the epithelium of the native trachea of these two patients; nor were they found in the bronchial epithelium of two sets of transplanted lungs without evidence of rejection. Only rare Leu-7 positive lymphocytes were evident in the epithelium (0 to 2 cells/section) and submucosal glands (0 to 1 cell/section) of 20 lungs from autopsied patients who had not received a transplant. Application of this technique to epithelial biopsy specimens obtained at bronchoscopic examinations demonstrated that it could be applied to the diagnosis of rejection in living heart-lung transplant recipients.