Neurokinin A is the predominant tachykinin in human bronchoalveolar lavage fluid in normal and asthmatic subjects

BACKGROUND—Multiple sensory neuropeptides are present in human airways and may contribute to diseases such as asthma. This study quantified and characterised substance P (SP), neurokinin A (NKA), and calcitonin gene related peptide (CGRP) immunoreactivity in bronchoalveolar lavage fluid in asthmatic and normal subjects.
METHODS—Using specific radioimmunoassay (RIA), SP, NKA and CGRP were measured in bronchoalveolar lavage fluid from asthmatic subjects (n = 5), normal subjects (n = 5), atopic non-asthmatic subjects (n = 6), and asthmatic subjects four hours after allergen challenge (n = 12). Peptide immunoreactivity was characterised using high performance liquid chromatography (HPLC) and RIA.
RESULTS—No SP or CGRP immunoreactivity was detected in any of the fractions from samples after extraction, HPLC, and RIA. Non-specific binding resulted in spurious SP immunoreactivity being detected in bronchoalveolar lavage fluid when no extraction process was employed. NKA was detected in significant amounts in asthmatic (median 550, range 425-625 pg/ml) and normal subjects (median 725, range 350-1425 pg/ml). The level of NKA was significantly higher in the asthmatic subjects after allergen challenge (median 750, range 350-1250 pg/ml) than in unchallenged asthmatic subjects (median 600, range 425-600 pg/ml, p<0.01).
CONCLUSIONS—Extraction and characterisation of peptides from bronchoalveolar lavage fluid must be performed to ensure that the measured immunoreactivity represents target peptide. NKA is present in bronchoalveolar lavage fluid in high concentrations and is the predominant tachykinin. The concentrations of NKA are similar in normal subjects and subjects with mild asthma.

Histamine release from bronchoalveolar lavage cells from asthmatic subjects after allergen challenge and relationship to the late asthmatic response

Background


Metachromatic cells obtained from asthmatic subjects demonstrate increased spontaneous and stimulated histamine release in vitro. Their ability to synthesize and store proinflammatory cytokines has focused renewed interest on their role in asthma.


Objective: The late asthmatic response provides a useful model of clinical asthma. The aim of the study was to examine metachromatic cell derived mediators and histamine releasability in vitro after in vivo allergen exposure in atopic subjects with and without asthma and relate them to the type of physiological response observed.

Methods: Bronchoalveolar lavage (BAL) cells were obtained 4 h after challenge from asthmatics exhibiting a single early response (EAR, n = 5), a dual response (LAR, n = 7), unchallenged (basal, n = 5), atopic non-asthmatic (ANA, n = 6) and non-atopic non-asthmatics (normal, n = 5). BAL histamine and tryptase concentrations and in vitro histamine release (HR) after stimulation with anti-IgE, allergen, A23187, conconavalin A and substance P were compared.

Results:Metachromatic cell numbers were lower in normal controls compared with all asthmatic groups and in LAR compared with EAR. Metachromatic cell derived mediators were higher in asthmatic compared with normal subjects. Spontaneous HR in LAR (20.5 ± 5.0%) was lower than EAR (29.5 ± 3.9%) and ANA (30.2 ± 1.4%) (P < 0.05). No differences were seen in stimulated HR between EAR and LAR. HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). HR in ANA stimulated with anti-IgE was greater than LAR (P < 0.05). After stimulation with ionophore A23187 (1 μM), release was greater in LAR compared with basal (P < 0.05) and no different at 5 μM. All subject groups responded to substance P (SP) but was significantly more in the asthmatic subjects compared to normal controls (P < 0.05). Allergen challenge did not modify the response of asthmatic subjects to SP.

Conclusion: Functional differences in metachromatic cell reactivity are present in atopic subjects 4 h after in vivo allergen exposure which relate to the physiological response observed after this time and suggest that there is ongoing metachromatic cell degranulation subjects who subsequently develop LAR.

Investigating paediatric airways by non-bronchoscopic lavage: Normal cellular data

Background: Bronchoscopic bronchoalveolar lavage in children to investigate bronchia disorders such as asthtna has both ethical and procedural difficulties.


Objective: The aim of this study was to establish a standardized non-bronchoscopic method to perform bronchoalveolar lavage in children attending for elective surgery to obtain normal cellular data.


Methods: Bronchoalveolar lavage was performed on normal children (n= 55) by infusing saline (20 mL) through an 8 FG suction catheter passed after endotracheal intubation. Oxygen saturation, heart and respiratory rate were monitored during the bronchoalveolar lavage procedure. Cellular analysis and total protein estimation of the lavage fluid were performed. Epithelial lining fluid volume was calculated (n = 15) using the urea dilution method.


Results: The procedure was well tolerated by all children. Total cell count and differential cell count for children (macrophages 70.8 ± 2.3%, lymphocytes 3.8 ± 0.6%, neutrophils 5,7 ± 1.0%, eosinophils 0.14 ± 0.03%. epithelial cells 19.6 ± 2.1%, mast cells 0.21 ± 0.02%) were similar to those reported for adults. Age and sex comparisons revealed no differences between groups. The mean total protein recovered in the cell free supernatant was 49.72 ± 4.29 mg/L and epithelial lining fluid volume was 0.82 ± 0.11% of return lavageate.


Conclusion This method allows bronchoalveolar lavage to be performed safely and quickly on children attending for routine elective surgery. Using this method and taking the ‘window of opportunity’ of elective surgery, the presence or absence of airway inflammation could be studied in children with various patterns of asthma during relatively asymptomatic periods.

Bronchoalveolar lavage findings suggest two different forms of childhood asthma

Background: It seems plausible that children with atopy and persistent asthma symptoms will, like their adult counterparts, have chronic airways inflammation. However, many young children with no other atopic features have episodic wheezing that is triggered solely by viral respiratory infections. Little is known as to whether airways inflammation occurs in these two asthma patterns during relatively asymptomatic periods.

Methods: Using a non-bronchoscopic bronchoalveolar lavage (BAL) procedure on children presenting for an elective surgical procedure, this study has investigated the cellular constituents of BAL fluid in children with a history of atopic asthma (AA) non-asthmatic atopic children (NAA) or viral associated wheeze (VAW).

Results: A total of 95 children was studied: 52 with atopic asthma (8.0 years, range 1.1-15.3, 36 male), 23 with non-asthmatic atopy (median age 8.3 years, range 1.7-13.6, 11 male) and 20 with VAW (3.1 years, range 1.0-8.2, 13 male). No complications were observed during the lavage procedure and no adverse events were noted post-operatively. Total lavage fluid recovered was similar in all groups and the total cell numbers were higher in the VAW group. Eosinophil (P< 0.005) and mast cell (/'<0.05) numbers were significantly elevated in the group with atopic asthma.

Conclusions: During relatively asymptomatic periods there is on-going airways inflammation, as demonstrated by eosinophil and mast cell recruitment, in children with asthma and atopy but not in children with viral associated wheeze or atopy alone. This strongly suggests that there are different underlying pathophysiologicai mechanisms in these two groups of children who wheeze.

Substance P induces histamine release from human pulmonary mast cells.

Substance P elicits histamine release from human skin and rodent mast cells. Since neuropeptide-mediated reflexes may be important in asthma, we examined the ability of substance P to stimulate human mast cells obtained at bronchoalveolar lavage (BAL). BAL samples were obtained at routine bronchoscopy from 35 non-preselected patients. Histamine release experiments were performed in a standard manner using substance P and the calcium ionophore A23187. Both substance P (50 μM) and A23187 caused histamine release (median 26.7%, range 6.2–62.8% and 32.1%, 7.7–56.8% respectively) which was significantly greater (P < 0.0001) than the spontaneous release (median 15.6%, range 4.1–33.4%), i.e. that in the absence of any stimulus. Substance P induced histamine release was via an energy dependent process and was blocked by preincubation with antimycin A. A significant correlation was observed between substance P induced release and spontaneous release but was not observed with A23187 induced release. Mast cell counts correlated significantly with substance P induced release but not with spontaneous or A23187 induced release. The substance P induced histamine secretion was elicited at similar concentrations to those used with rodent and human skin mast cells. Asthma is associated with increased numbers of mast cells which have both increased spontaneous and stimulated secretory responses. Thus, in vivo, the bronchoconstrictor action of substance P may in part result from activation of mast cells in the bronchial lumen.

Electronic cell counting to measure total cell numbers in bronchoalveolar lavage fluid

Cell counting of bronchoalveolar lavage (BAL) fluid is performed manually in routine practice. This has both methodological and inherent errors; however, the accuracy and suitability of automated counting devices have been questioned. In this study, a Coulter(R) Counter D Industrial model was calibrated and then used to measure the total cell count in unprocessed bronchoalveolar lavage fluid, and compared to a standard manual method.