It has been suggested that airway irritation, by acting as an adjuvant, as well as producing damage, may be an important factor related to asthma. The present study examined the window of time following acute upper and lower airway irritant exposure to determine the period of increased risk of immunological sensitization. Brown Norway rats were exposed to 87 ppm NO2 or 1000 ppm NH3 for 1 hr. A 30-min ovalbumin (OVA) exposure of 18.14 microg/liter air was given at various times based upon the time course of irritant associated inflammatory response (either immediately prior to or 1 or 7 days after the irritant exposure). OVA-only, NO2-only or NH3-only controls, and saline controls were also studied. Weekly booster exposures of OVA (or saline) were given. Circulating OVA-specific IgE, IgA, and IgG levels were quantified periodically during the 6 weeks of the study. Bronchoalveolar lavage (BAL) was also performed to examine the inflammatory response to allergic and irritant challenge. Significant increases in OVA-specific IgE, IgG, and IgA antibody titers were seen in rats given the sensitizing OVA exposure within 1 day of the NO2, but not NH3 exposures. Enhancement of cellular infiltrate in BAL was noted in groups given the sensitizing OVA exposure within 1 day of the NO2 or NH3. It is concluded that the inflammatory and immunological response to antigen exposure can be modified by the site of respiratory tract irritation and the relative times of irritant and antigen exposure.
Late allergic airway responses can be transferred by CD4+ T cells in the rat. To investigate the role of T-cell cytokines in these responses, we examined the expression of mRNA for Th2 (interleukin [IL]-4 and IL-5) and Th1 (IL-2 and interferon gamma [INF-gamma])-type cytokines in Brown Norway rats that were administered either antigen-primed W3/25(CD4)+ or OX8(CD8)+ T cells. Donors were actively sensitized by subcutaneous injection of ovalbumin (OVA) in the neck and T cells were obtained from the cervical lymph nodes by immunomagnetic cell sorting for administration to unsensitized rats. Control rats received bovine serum albumin (BSA)-primed CD4+ and CD8+ T cells. Two days later, recipient rats were challenged with aerosolized OVA, and bronchoalveolar lavage (BAL) was performed 8 h after challenge. BAL cells expressing mRNA for IL-2, IL-4, IL-5, and INF-gamma were analyzed using the technique of in situ hybridization. Recipients of OVA-primed CD4+ T cells had an increase in the fraction of BAL cells expressing mRNA for IL-4 and IL-5 compared with BSA-primed CD4+ or OVA-primed CD8+ cells (P
To evaluate the role of lymphocytes in the pathogenesis of allergic bronchoconstriction, we investigated whether allergic airway responses are adoptively transferred by antigen-primed lymphocytes in Brown Norway (BN) rats. Animals were actively sensitized to ovalbumin (OA) or sham sensitized, and 14 d later mononuclear cells (MNCs) were isolated from intrathoracic lymph nodes, passed through a nylon wool column, and transferred to naive syngeneic rats. Recipients were challenged with aerosolized OA or bovine serum albumin (BSA) (5% wt/vol) and analyzed for changes in lung resistance (RL), airway responsiveness to inhaled methacholine (MCh), and bronchoalveolar lavage (BAL) cells. Recipients of MNCs from sensitized rats responded to OA inhalation and exhibited sustained increases in RL throughout the 8-h observation period, but without usual early airway responses. Recipients of sham-sensitized MNCs or BSA-challenged recipients failed to respond to antigen challenge. At 32 h after OA exposure, airway responsiveness to MCh was increased in four of seven rats that had received sensitized MNCs (p = 0.035). BAL eosinophils increased at 32 h in the recipients of both sensitized and sham-sensitized MNCs. However, eosinophil numbers in BAL were inversely correlated with airway responsiveness in the recipients of sensitized MNCs (r = -0.788, p = 0.036). OA-specific immunoglobulin E (IgE) was undetectable by enzyme-linked immunosorbent assay (ELISA) or passive cutaneous anaphylaxis (PCA) in recipient rats following adoptive transfer. In conclusion, allergic late airway responses (LAR) and cholinergic airway hyperresponsiveness, but not antigen-specific IgE and early responses, were adoptively transferred by antigen-primed lymphocytes in BN rats.(ABSTRACT TRUNCATED AT 250 WORDS)
BACKGROUND: Macrophages are involved in asthma, but their pulmonary turnover is unknown. We compared the ability of bronchoalveolar lavage (BAL) and bronchial macrophages to proliferate in normal subjects and patients with asthma. METHODS: BAL cells from eight patients with asthma and eight normal volunteers were separated with a discontinuous Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden). In a first experiment, nuclei of each alveolar macrophage (AM) fraction, stained with propidium iodide, were analyzed for DNA content with a flow cytometer, and the proportions of cells in the G0/G1, S, and G2 + M phases were determined. In a second experiment, expression of Ki-67-related antigen was sought on AMs by immunocytochemistry. Macrophages from 10 patients with asthma and 10 normal volunteers were studied in biopsy specimens by means of immunohistochemistry with a panmacrophage monoclonal antibody (HAM-56) and a monoclonal antibody against proliferating cell nuclear antigen. RESULTS: The proportions of BAL AMs in the different phases of the cell cycle were similar in normal subjects and patients with asthma for all fractions, and the percentage of cells in S and G2 +/- M phases ranged from 7.3% to 11.3%. Under 1% of BAL AMs expressed Ki-67-related antigen. None of the macrophages present in the biopsy specimens expressed proliferating cell nuclear antigen. CONCLUSIONS: This study does not indicate that an important source of airway macrophages is local proliferation.
In vitro and in vivo anti-inflammatory properties and soft characteristics of etiprednol dicloacetate (BNP-166) a new steroid, which has been developed for the treatment of asthma, were investigated in this study. The compound effectively decreased cytokine production in lipopolysaccharide stimulated lymphocytes and attenuated lectin-induced proliferation of blood mononuclear cells in tissue culture. In an animal model of allergen sensitized and challenged Brown Norway rats, using topical treatment, etiprednol dicloacetate substantially attenuated the extent of allergen induced bronchoalveolar fluid eosinophilia. At every examined parameter its pharmacological effects were comparable to those of budesonide. By means of in vitro biological and analytical methods the soft character of BNP-166 was also investigated. The anti-inflammatory effect of etiprednol dicloacetate in vitro was shown to be the function of the quantity of serum components, present in the assay. This loss of activity was most likely the result of the fast metabolism of etiprednol dicloacetate, which in the presence of sera could have been demonstrated by LC/MS/MS. Our data indicate that the significant local effect of the compound will very likely be accompanied with a drastically reduced systemic activity indicating an encouraging selectivity of the pharmacological action of etiprednol dicloacetate.
Viral respiratory infections cause acute airway abnormalities consisting of inflammation and physiological dysfunction in both animals and humans. It is likely that inflammatory cell products, such as cytokines, contribute substantially to viral-induced airway dysfunction. We hypothesized that imiquimod, an immune response enhancing agent that induces interferon-alpha, would attenuate the development of airway dysfunction during acute viral illness in rats. Adult Brown Norway rats were inoculated with parainfluenza type 1 (Sendai) virus or sterile vehicle, and treated with either imiquimod or water. Respiratory system resistance (Rrs), arterial oxygen tension (Pa,O2), lung viral titres and bronchoalveolar lavage (BAL) leucocyte counts were measured in anaesthetized, paralysed, ventilated rats. Virus-infected, water-treated rats had a significant decrease in Pa,O2 and had significant increases in leucocyte count and Rrs when compared to both the virus-infected, imiquimod-treated, (Pa,O2, p = 0.03; leucocyte count, p = 0.02; and Rrs, p = 0.009) and noninfected, water-treated rats (Pa,O2, p = 0.007; leucocyte count, p = 0.001; and Rrs, p = 0.01). In addition, imiquimod suppressed BAL eosinophils in both virus-infected (p = 0.02) and noninfected (p = 0.001) groups, and lowered overall virus titres (p = 0.03). Thus, both virus-induced airway inflammation and physiological dysfunction were attenuated significantly by imiquimod treatment in this animal model. By further delineating mechanisms by which infections induce airway dysfunction in animal models, more specific pharmacological interventions can be developed for the treatment of virus-induced asthma.
Asthma is considered to be a chronic inflammatory response of the airways characterized by a leukocyte infiltration into the lungs. Whereas lymphocytes and macrophages are involved in the initiation and propagation of inflammation, both neutrophils and in particular eosinophils are considered to play major effector roles. Therefore, allergic animal models in various species have been established to assess leukocyte infiltration by bronchoalveolar lavage (BAL) of antigen-sensitized and antigen-challenged animals as an inflammatory parameter in asthma pharmacology. Differential leukocyte counts in BAL fluids are routinely assessed by visual microscopic analysis of stained slides after cytocentrifugation. This procedure is very time-consuming, and the underlying standard morphological criteria may vary between different observers. In the present paper, we propose an alternative automatic method for leukocyte differentiation in BAL fluids from ovalbumin-treated guinea pigs and Brown-Norway rats using Cobas Helios 5Diff from Hoffmann-La Roche. BAL samples are directly applied to the analyzer and are automatically mixed with "Eosinofix," which stabilizes leukocyte membranes and specifically stains eosinophils. By a combination of electric (resistance) and optical (light scatter) analysis, the lymphocytes, monocytes/macrophages, neutrophils, and eosinophils are discriminated and the total leukocyte numbers are obtained. For both animal species we found high correlations for all leukocyte populations by comparing the results obtained with Cobas Helios 5Diff and conventional microscopic analysis. The major advantage of the automatic method is the much lower (about one-third) time requirement.
One of objective methods of early and differential diagnosis of occupational pulmonary diseases in miners (pneumoconiosis, silicotuberculosis, dust bronchitis) is bronchoscopy with a cytologic examination of bronchoalveolar lavage fluid (BAF). BAF-examination was carried out in a total of 88 patients with incipient and advanced forms of dust bronchitis, pneumoconiosis and silicotuberculosis. A direct relationship has been revealed between a decline in local cell-bound immunity caused by a dust-inducted affection mononuclear phagocytes and advancing of stages of dust-related diseases.
The pathogenesis of parainfluenza 1 (Sendai) virus infection was compared among 25-day-old BN, F344, and LEW rats to identify a sensitive as well as a resistant inbred rat strain to Sendai virus-induced lung injury during early life. At 7 days after inoculation, BN rats had 65-fold higher (P less than .001) pulmonary viral titers and threefold higher (P less than .002) numbers of neutrophils in bronchoalveolar lavage fluid than did F344 rats. At 14 days after inoculation, when most virus-induced inflammation had been resolved, BN rats had a threefold greater (P less than .01) incidence of bronchioles with aggregates of lymphocytes and macrophages than did F344 rats. Control BN rats had higher numbers of bronchiolar eosinophils than did F344 or LEW rats. Although viral inoculation resulted in increased numbers of bronchiolar mast cells in all three strains at 14 days, bronchiolar mast cell density was greater (P less than .005) in virus-inoculated BN and LEW rats than in F344 rats. We conclude that BN rats are high responders and F344 rats are low responders to Sendai virus-induced bronchiolitis, pneumonia, and airway mastocytosis. These strain differences may be useful in elucidating important pathogenetic mechanisms in virus-induced airway injury and mastocytosis.
To test the hypothesis that CD8+ T cells may suppress the allergen-induced late airway response (LAR) and airway eosinophilia, we examined the effect of administration of Ag-primed CD8+ T cells on allergic airway responses, bronchoalveolar lavage (BAL) leukocytes, and mRNA expression for cytokines (IL-4, IL-5, and IFN-gamma) in OVA-sensitized Brown Norway rats. On day 12 postsensitization to OVA, test rats were administered 2 million CD8+ T cells i.p. isolated from either the cervical lymph nodes (LN group; n = 8) or the spleen (Spl group; n = 6) of sensitized donors. On day 14, test rats were challenged with aerosolized OVA. Control rats were administered PBS i.p. on day 12, and challenged with OVA (n = 10) or BSA (n = 6) on day 14. The lung resistance was measured for 8 h after challenge. BAL was performed at 8 h. Cytospin slides of BAL were analyzed for major basic protein by immunostaining and for cytokine mRNA by in situ hybridization. The LAR was significantly less in the LN group (1.8 +/- 0.5 U; p