Effects of aging on surfactant forms in rats.

Surfactant present in the alveolar space exists in two major forms: functional large aggregate forms (LA) and nonfunctional small aggregate forms (SA), but there is no information about the changes of surfactant forms and the rate of conversion of LA to SA in the aged lungs. The purpose of the present study was to investigate the developmental aspects of surfactant forms in newborn, young, middle-aged and aged rats, LA and SA were recovered from alveolar lavages of rats. The rate of conversion from LA to SA was then analysed using a surface-area cycling technique. Age-related changes of saturated phosphatidylcholine (Sat-PC) and surfactant protein A (SP-A) pool sizes were also evaluated in alveolar lavages. The alveolar lavages recovered from aged rats contained a significantly higher proportion of LA than did those obtained from young or newborn rats. There was also an age-related decrease in the rate of conversion from LA to SA in vitro. The Sat-PC pool sizes in the alveolar lavages decreased with age, but the SP-A contents were similar between young and aged rats. These results suggested that decreased form conversion may contribute to maintaining functional surfactant pool sizes in the lungs of aged rats.

Increased levels of surfactant protein A and D in bronchoalveolar lavage fluids in patients with bronchial asthma.

Surfactant proteins (SP)-A and SP-D are collagen-like glycoproteins belonging to the "collectin" class of C-type lectins, which are primarily synthesized in type II cells. Recent studies reported the possibility of local production of SP-A and SP-D in the airways, but the amounts of surfactant proteins in patients with bronchial asthma have not been studied. The composition of surfactant proteins in mild, stable asthmatics in the first lavage as bronchial lavage (BL) and the second and third lavages consecutively as alveolar lavages (AL) were therefore, analysed separately. The co-relationships in the BL between the amounts of surfactant proteins and those of fucose, which is one of the markers of submucosal secretion were also analysed. Increased amounts of SP-A in BL and AL of in asthmatics were found as compared with those in controls. A high concentration of SP-D in the AL asthma patients was also found. The levels of SP-A correlated with those of fucose in patients with bronchial asthma (r=0.849, p<0.01). The observations in the present study suggested that surfactant protein A may be secreted from the airways with allergic inflammation in a different manner from the alveoli. The increased levels of surfactant proteins A and D may play a protective role in an allergic inflammation in the pathogenesis of bronchial asthma.

Expression of interleukin-16 by human epithelial cells. Inhibition by dexamethasone.

Production of chemoattractants by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma and other pulmonary diseases. Recently, interleukin (IL)-16 (lymphocyte chemoattractant factor) was reported to be a potent chemotactic stimulus for CD4(+) T lymphocytes and eosinophils, the types of leukocyte found in the proximity of bronchial epithelium in asthmatic individuals. To test the possibility that bronchial epithelial cells produce IL-16, we analyzed RNA and culture supernatants from the human bronchial epithelial cell line BEAS-2B, using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. BEAS-2B constitutively expressed IL-16 messenger RNA (mRNA) and protein; IL-16 expression was significantly upregulated in a concentration-dependent manner within 24 h by stimulation with histamine, IL-1beta, or tumor necrosis factor (TNF)-alpha whereas interferon-gamma did not significantly increase IL-16. Findings in BEAS-2B cells were confirmed in primary bronchial epithelial cells. Using TA cloning, IL-16 was cloned from BEAS-2B airway epithelial cells. Sequence analysis confirmed its near identity with lymphocyte-derived IL-16. The combination of IL-1beta and TNF-alpha had an additive effect on IL-16 expression. This combination of cytokines also had a priming effect on histamine-induced IL-16 mRNA expression, which was observed within 24 h and which increased to at least 48 h after stimulation. The IL-16 expression induced by histamine and combined cytokines was significantly inhibited by pretreatment with the protein synthesis inhibitor cycloheximide (10 microg/ml). Pretreatment with dexamethasone also significantly suppressed the expression of IL-16, in a concentration-dependent manner. Sputum samples from asthmatic subjects were found to have higher levels of IL-16 than were samples from subjects with other pulmonary inflammatory diseases. These findings suggest that bronchial epithelial cells have the capacity to produce IL-16 after stimulation with histamine, IL-1beta, and TNF-alpha, and raise the possibility that epithelium-derived IL-16 may play a role in recruitment of eosinophils and CD4(+) T lymphocytes in the airways. Downregulation of IL-16 expression by dexamethasone suggests that glucocorticoids may inhibit airway inflammation partly by suppressing the synthesis of inflammatory cytokines including IL-16.

Suppressive effects of SP-A on ionomycin-induced IL-8 production and release by eosinophils.

Recent studies have demonstrated that pulmonary surfactant protein (SP)-A plays a potential role in modifying inflammation and immune function. To see whether SP-A could modify IL-8 production and release by eosinophils stimulated with ionomycin, SP-A purified from surfactant recovered from patients with alveolar proteinosis was added to eosinophils isolated by the negative-selection method with immunomagnetic beads, and cultured for 24 h. The concentrations of IL-8 in the cell-free supernatants and cell lysates were then measured by ELISA. SP-A attenuated the production of IL-8 by eosinophils in a concentration-dependent manner. SP-A also attenuated the release of IL-8 from the eosinophils. The addition of SP-A antibody (PE10) reversed these effects of SP-A completely. These data suggest that SP-A may have the potential to modify allergic inflammation by inhibiting the release and production of IL-8 by eosinophils.

Serum levels of eosinophil cationic protein and IL-5 in patients with asthma without systemic corticosteroids.

We measured the serum levels of eosinophil cationic protein (ECP) and interleukin-5 (IL-5) in 39 patients with asthma (symptomatic 12, asymtomatic 27) without systemic corticosteroids and 49 healthy subjects. There were significant differences in serum levels in both ECP and IL-5 between symptomatic and asymptomatic asthma, and healthy subjects. A significant positive correlation was found between serum levels of ECP and IL-5. No significant correlation was found between %FEV1 and serum level of ECP or IL-5, however, when the analysis was restricted to patients of less than 60 years of age, the correlations were significant. These results suggest that IL-5 is one of the factors that activate eosinophils even in peripheral blood, and that measurement of serum levels of ECP and IL-5 is useful for in vitro monitoring of asthma.

[A case with systemic lupus erythematosus complicated with tuberculosis sacroiliac arthritis].

A 26-year-old woman was admitted to our hospital because of fever of unknown origin. She had been treated with prednisolone, elcatonin and alfacalcidol under the diagnosis of systemic lupus erythematosus (SLE) and aseptic necrosis of femoral bone head. Six months ago she began to have a fever and subsequently left low back pain, for which extensive examinations were performed in other hospital but their causes remained unclear. She was referred to our hospital for further evaluation and therapy in October 1995. Bacteriological, immunological or serological examinations did not reveal the origin of fever. CT and ultrasonic examination did not show any abnormality. However, MRI, which was taken for the evaluation of aseptic necrosis of femoral bone head, showed the abscess shadow in sacroiliac joint. Open biopsy was performed and Mycobacterium tuberculosis bacilli were detected from the abscess. To our best knowledge, this is the first report of SEE with tuberculosis sacroiliac arthritis.

Eosinophil apoptosis caused by theophylline, glucocorticoids, and macrolides after stimulation with IL-5.

BACKGROUND: Glucocorticoids have long been used as the most potent drugs in the treatment of bronchial asthma. Data reported recently have led to the proposal that theophylline and macrolides have antiinflammatory effects. OBJECTIVE: We examined the abilities of theophylline, glucocorticoids, and macrolides to counteract the prolongation of eosinophil survival caused by IL-5. METHODS: Purified guinea pig eosinophils were cultured in the presence or absence of human IL-5 and with or without the aforementioned drugs at various concentrations. The percentage of cells alive after 3 days in culture was determined. RESULTS: Aminophylline (AM), methylprednisolone (MP), erythromycin (EM), and clarithromycin (CAM) suppressed the IL-5 induced prolongation of eosinophil survival in a dose-dependent manner. The effects of these drugs on eosinophil survival were significantly greater at low concentrations of IL-5 than at high concentrations of IL-5. When eosinophils were cultured in the presence of IL-5 (1 ng/ml) with physiologic concentrations of MP (10(-6) mol/L), AM (10(-4) mol/L), and either EM or CAM (both 10 micrograms/ml), the effect of IL-5 was almost completely abolished, and the morphologic changes in eosinophils observed by electron microscopy were consistent with apoptosis. DNA extracted from eosinophils cultured with IL-5 and each of the drugs was definitely fragmented. CONCLUSIONS: One mechanism of the effectiveness of these drugs is induction of eosinophil apoptosis. Some combination of these drugs may be useful in the treatment of bronchial asthma.

Eosinophil peroxidase stimulates the release of granulocyte-macrophage colony-stimulating factor from bronchial epithelial cells.

BACKGROUND: Asthma is characterized by an accumulation of activated eosinophils in the airway. Eosinophil viability-enhancing activity is present in the sputum of patients with asthma, largely because of granulocyte-macrophage colony-stimulating factor (GM-CSF). Bronchial epithelial cells have been shown to release cytokines including GM-CSF when stimulated with IL-1 beta or tumor necrosis factor-alpha. OBJECTIVE: The study was designed to determine whether eosinophil peroxidase (EPO) stimulates the release of GM-CSF from bronchial epithelial cells. METHODS: Epithelial cells (BEAS-2B) were cultured in serum free HD-F12 medium in a 24-well tissue culture plate until they became confluent. The cells were then exposed to EPO (5.9 x 10(-8) to 5.9 x 10(-7) mol/L) for 15 minutes, washed twice, and cultured in 1 ml of HD-F12. The supernatants were harvested at 3, 6, or 24 hours, and GM-CSF concentration was measured by ELISA. BEAS-2B cells were also treated with a system comprising EPO (1.9 x 10(-9) to 5.9 x 10(-8) mol/L) + 10(-5) mol/L H2O2 + 10(-4) mol/L Br for 24 hours. RESULTS: The GM-CSF concentration in the supernatant pretreated with EPO increased in a time- and concentration-dependent manner compared with control. The release of GM-CSF was not inhibited by catalase but was inhibited by cyclohexamide and by mixing of EPO with heparin, suggesting that the action is due to the cationic property of EPO. When EPO was combined with H2O2 and Br, 5.9 x 10(-9) mol/L EPO + 10(-5) mol/L H2O2 released two times more GM-CSF into the supernatants compared with control. CONCLUSION: These results suggest that EPO stimulates epithelial cells to release GM-CSF and forms a self-stimulatory cycle.

Comparison between allergen-induced and exercise-induced asthma with respect to the late asthmatic response, airway responsiveness, and Creola bodies in sputum.

BACKGROUND: The difference between allergen-induced asthma and exercise-induced asthma with respect to the late asthmatic response and airway responsiveness has not been well elucidated. OBJECTIVE: We compared the incidence of late asthmatic response, the changes in airway responsiveness, the degree of epithelial desquamation, and the activation of eosinophils in the airways after induction of allergen-induced asthma and exercise-induced asthma. METHODS: Allergen-induced asthma or exercise-induced asthma was provoked in asthmatic patients, and sputum was collected before challenge and at the immediate asthmatic response and the late asthmatic response. Clusters of columnar epithelial cells in sputum (Creola bodies) were detected to evaluate respiratory epithelial damage, and the sputum eosinophil cationic protein (ECP) concentration was measured to evaluate eosinophil activation in the airways. Airway responsiveness was measured before and 48 hours after the challenge. RESULTS: The maximal % fall in FEV1 with the late asthmatic response was significantly higher after induction of allergen-induced asthma than after exercise-induced asthma, even though the maximal % fall in FEV1 with the immediate asthmatic response was similar. Airway responsiveness increased significantly at 48 hours after allergen-induced asthma, while it did not change after exercise-induced asthma. The increase in airway responsiveness was not correlated with the maximal % fall in FEV1 with the late asthmatic response, but was correlated with the degree of epithelial damage evaluated by observation of Creola bodies. The sputum ECP concentration and the percentage of sputum eosinophils increased significantly with the late asthmatic response after allergen-induced asthma, but did not change after exercise-induced asthma. CONCLUSIONS: We conclude that less airway inflammation was provoked by exercise-induced asthma resulting in less epithelial damage and no increase of airway responsiveness in contrast to allergen-induced asthma.

Damage to respiratory epithelium by guinea-pig eosinophils stimulated with IgG-coated Sepharose beads.

BACKGROUND: There are no reports of respiratory epithelial damage induced by immunoglobulin-stimulated eosinophils. OBJECTIVE: We tried to induce damage to respiratory epithelium by guinea-pig (GP) eosinophils stimulated with guinea-pig IgG (GP-IgG)-coated Sepharose 4B beads. METHODS: GP tracheal epithelium was cultured together with GP eosinophils that had been collected from the peritoneal cavity, purified on a Percoll gradient, and stimulated with GP-IgG-coated beads (GP eosinophils:beads = 20:1). Damage to the epithelium was observed with an inverted microscope. RESULTS: After 24 h of culture with three 10(6) eosinophils, irregularity of the surface of the epithelium, desquamation, shedding of cilia, and abnormal beating of cilia were observed. This damage was first observed after 12 h of incubation, and was more severe at 24 h. No damage was found when beads coated with human serum albumin (HSA) were used. GP eosinophils stimulated with GP-IgG released significantly more EPO than those stimulated with HSA, at 6 and 24 h. Heparin and catalase partially inhibited the epithelial damage. O2-production by eosinophils was also enhanced with GP-IgG-coated beads. CONCLUSION: Both granule basic proteins and reactive oxygen radical may be responsible for epithelial damage, probably via an EPO + H2O2 + halide system. These results confirmed that stimulated eosinophils can damage respiratory epithelium.

Detection of transforming growth factor-beta in sputum from patients with bronchial asthma by eosinophil survival assay and enzyme-linked immunosorbent assay.

BACKGROUND: We have shown that interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are present in sputum from patients experiencing acute asthma attacks, by eosinophil survival assay. The viability of guinea-pig eosinophils was significantly increased in the presence of such sputum extracts after 3 days' culture, and it was inhibited by the addition of anti-IL-5 and anti-GM-CSF antibodies. However, the contribution of IL-5 to the increase in eosinophil viability was less than expected from the values of IL-5 measured by enzyme-linked immunosorbent assay (ELISA). Therefore, we speculated that something in sputum inhibited the function of IL-5. OBJECTIVE: Transforming growth factor-beta (TGF-beta) was the only cytokine we tested that inhibited the prolongation of survival of guinea-pig eosinophils induced by IL-5. The objective of this study is to detect TGF-beta in the same sputum. METHODS: Guinea-pig eosinophils were cultured with or without anti-TGF-beta antibody in the presence of sputum extracts, and the eosinophil viability was counted after 3 days. Measurement of TGF-beta 1 in sputum was performed by ELISA. RESULTS: Eosinophil viabilities with and without anti-TGF-beta antibody were 79.7 +/- 2.9% and 69.0 +/- 2.7%, respectively, and the difference between them was statistically significant (P < 0.05, n = 9). The concentration of TGF-beta 1 in the sputum was 21.7 +/- 3.3 ng/mL (n = 9). CONCLUSION: These observations suggest that TGF-beta is present in sputum from patients with bronchial asthma.

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.

[Measurement of serum and sputum eosinophil cationic protein concentrations in asthma].

Concentrations of eosinophil cationic protein (ECP) were measured in serum from patients with asthma, patients with chronic obstructive pulmonary disease (COPD), and healthy subjects. The relationships between serum ECP concentration and percent of predicted FEV1 (%FEV1), and between serum and sputum ECP concentrations were also examined in patients with asthma. Serum ECP concentration in asthma was significantly higher than those in COPD patients and healthy subjects. There was a significant inverse correlation between serum ECP concentration and %FEV1 when analysis was restricted to data from asthma patients less than 60 years old. ECP concentration in serum and in sputum were significantly correlated. These results suggest that in asthma patients, eosinophils are activated in serum, and that the degree of eosinophil activation in the airway can be estimated by measuring serum ECP to some extent. Because it does not require bronchial biopsy or bronchoalveolar lavage, measurement of serum ECP may be useful as a minimally invasive monitoring of asthma.

[A case of lupus cystitis successfully treated with corticosteroid accompanied by gastrointestinal symptoms].

A 34-years-old woman was admitted to our department in February, 1992, because of nausea, vomiting, abdominal pain and diarrhea. She had been diagnosed as systemic lupus erythematosus (SLE) in 1988 and treated with prednisolone at the dose of 5 mg a day. In December, 1991, gastrointestinal symptoms developed followed by anuria on March 3, 1992. The laboratory findings revealed no activities in SLE. Computed tomography (CT) showed bilateral hydroureteronephrosis, swelling of bladder and gastrointestinal wall, and ascites. Under the diagnosis of lupus cystitis, corticosteroid therapy was started with 125 mg of methylprednisolone. Her symptoms improved immediately. Abnormal findings shown in the previous CT disappeared concomitantly. Lupus cystitis was reported by Orth et al. 1983 as severe fetal syndrome. However, because early corticosteroid therapy appears to reverse acute manifestation of lupus cystitis without complications, attention should be paid on lupus cystitis in patients with SLE with gastrointestinal symptoms of unknown etiology and decreasing urinary volume.

Inhibition of leukotriene C4 and B4 release by human eosinophils with the new 5-lipoxygenase inhibitor 6-hydroxy-2(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride.

Eosinophils generate and release leukotrienes C4 and B4 (LTC4, LTB4) and platelet activating factor (PAF), all of which have the capacity to cause inflammation and tissue injury in the airways. This study has examined the effects of a new 5-lipoxygenase inhibitor, 6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiazo le hydrochloride (CAS 120164-49-0, E6080) on the release of LTC4, LTB4 and PAF by human eosinophils, Eosinophils stimulated by 1 mumol/l calcium ionophore A23187 for 15 min released 37.5 +/- 2.2 ng, 2.3 +/- 0.3 ng and 4.0 +/- 0.3 pmol per 10(6) cells of immunoreactive LTC4, LTB4 and PAF, respectively (mean +/- SEM, n = 4). LTC4 and LTB4 releases were inhibited dose-dependently by the addition of E6080 to the cell suspension. The IC50 values were 0.26 mumol/l for LTC4 and 0.23 mumol/l for LTB4. PAF release was not inhibited. These results suggest that E6080 is a potent inhibitor of LTC4 and LTB4 release from eosinophils and may provide a protective effect against bronchoconstriction during late-phase asthmatic responses.

Cyclosporine A reduces T lymphocyte activity and improves airway hyperresponsiveness in corticosteroid-dependent chronic severe asthma.

BACKGROUND: Although cyclosporine A is proving effective for chronic severe asthma, its mechanism of action in this disease is unclear. OBJECTIVE: This open study was conducted to determine whether cyclosporine A therapy would reduce the degree of airway hyperresponsiveness and T lymphocyte activity. METHODS: After a 6-week run-in period, nine patients with corticosteroid-dependent chronic severe asthma were treated with cyclosporine A (initial dose 5 mg/kg per day) for 12 weeks. RESULTS: Weekly mean morning peak expiratory flow significantly increased in six subjects during the last 6 weeks of trial. Geographic mean PC20-acetylcholine (the provocative concentration of acetylcholine required to cause a 20% fall in FEV1) was 0.147 mg/mL before cyclosporine A treatment and increased to 0.216 mg/mL at 6 weeks and to 0.379 mg/mL at 12 weeks after treatment. The increase at 12 weeks was statistically significant (P < .05). The percentage of CD4-positive T lymphocytes bearing IL-2 receptor (a marker of T cell activation) in the peripheral blood decreased significantly at 6 weeks (P < .05), but returned to baseline value at 12 weeks, probably due to cyclosporine A dose reduction in seven subjects. Serum IgE levels and peripheral blood eosinophil counts, however, which are dependent on IL-4 and IL-5, respectively, were still significantly decreased at 12 weeks, suggesting lymphokine production remained suppressed even after cyclosporine A dose was reduced. CONCLUSION: Taken together, these data suggest that cyclosporine A may act in asthma, at least in part, by inhibition of T lymphocyte activation and by reducing the degree of airway hyperresponsiveness.

Eosinophil viability-enhancing activity in sputum from patients with bronchial asthma, Contributions of interleukin-5 and granulocyte/macrophage colony-stimulating factor.

To investigate the mechanisms of eosinophil activation in the airways of patients with asthma, we attempted to detect eosinophil-activating cytokines in sputum extracts obtained from asthmatic patients during acute attacks or in remission by eosinophil survival assay. Purified guinea pig eosinophils were cultured in the presence or absence of sputum extracts, and the eosinophil viability was measured on Day 4. Eosinophil viability in the presence of sputum extracts derived from patients during moderate or severe attacks was significantly higher than that for sputum obtained from patients in remission or during mild attacks or from those with other respiratory diseases, including bronchiectasis and diffuse panbronchiolitis (p < 0.05). The total symptom score during the week prior to sputum collection correlated with the eosinophil viability (rs = 0.79, p < 0.01). Eosinophil viability-enhancing activity (EVEA) in the sputum of asthmatic patients with moderate or severe attacks was neutralized by anti-IL-5 antibody and by anti-GM-CSF antibody by 19.9 +/- 13.7% and 76.9 +/- 8.2% (mean +/- SEM, n = 7), respectively. EVEA was completely neutralized by a combination of anti-IL-5 and anti-GM-CSF antibodies. There was a significant correlation between the concentration of eosinophil cationic protein (ECP) in sputum extracts and the eosinophil viability (rs = 0.54, p < 0.05). These findings suggest that IL-5 and GM-CSF are present in the sputum during asthma attacks and that these cytokines are at least partially responsible for eosinophil activation in asthma.

Serum levels of soluble interleukin-2 receptor in asthma patients.

We measured the serum concentration of soluble interleukin-2 receptor (sIL-2R) in asthma patients and healthy controls to evaluate T-lymphocyte activation in this disease. The SIL-2R concentrations in patients with asthma irrespective of presence of acute attacks were significantly higher than those of the controls (p < 0.001). Although the sIL-2R concentrations were similar in the patients with acute asthma and those in remission, levels in patients with moderate and severe asthma attacks were significantly higher than levels for patients in remission (p < 0.001). These results suggest that T-lymphocyte activation occurs in asthma patients and becomes more prominent during moderate and severe acute attacks.

Detection of interleukin-5 messenger RNA and interleukin-5 protein in bronchial biopsies from asthma by nonradioactive in situ hybridization and immunohistochemistry.

Recently direct evidence for the role of interleukin-5 (IL-5) in eosinophilic inflammation in the airways of persons with asthma has been provided by an in situ hybridization study that used radioisotope-labeled IL-5 complementary RNA probes. Radioisotope-labeled probes, although sensitive, require autoradiographic detection, which is time-consuming. In the most recent study we attempted to detect IL-5 messenger RNA in the bronchial biopsy specimens from patients with asthma using nonradioactive in situ hybridization, which gives rapid results. Bronchial biopsy specimens were obtained from eight patients with asthma and seven diseased control subjects. IL-5 complementary DNA probes were labeled with digoxigenin-deoxyuridine triphosphate and hybridized to permeabilized sections. Hybridization signals were visualized by an immunohistochemistry technique. Positive hybridization signals were observed in six of the eight biopsy specimens from patients with asthma. Pretreatment with ribonuclease or hybridization with an unrelated probe produced negative results. Immunohistochemical staining of serial sections with a monoclonal antibody to IL-5 revealed that a few cells within the mucosa positively stained, suggesting active synthesis of IL-5. Biopsy results from the seven diseased control subjects did not show any hybridization signal. These results confirm and extend previous observations of IL-5 messenger RNA expression in the airways of patients with asthma, and suggest that digoxigenin-labeled IL-5 complementary DNA probes would be a powerful research tool.