Selective early increases of bronchoalveolar CD8+ lymphocytes in a LEW rat model of hypersensitivity pneumonitis.

BACKGROUND: The pathogenesis of hypersensitivity pneumonitis (HP) involves cell-mediated hypersensitivity; various bronchoalveolar T-cell subsets with uncertain roles in disease have been reported and implicated in the pathogenesis. OBJECTIVES: Previous studies at 72 hours after initial antigen challenges showed proportionate increases in T-cell phenotypes. Therefore we tested the hypothesis that early events in response to inhaled antigen in a LEW rat model of HP would include a disproportionate appearance in bronchoalveolar lavage fluid (BALF) and lung parenchyma of a specific T-effector cell responsible for subsequent inflammation and that these events could be identified by phenotyping. METHODS: We double labeled BALF and parenchymal lung lymphocytes with monoclonal antibodies and used flow cytometry to quantitate CD4+ and CD8+ phenotypic subsets 4 and 24 hours after inhalation of antigen. RESULTS: We found disproportionate increases in BALF CD8+ phenotypes. The strongest correlation with pathologic findings was for a putative cytotoxic effector (CD8+CD45R-) T lymphocyte. CONCLUSION: Meaningful interpretation of lung T-cell phenotype quantitation requires studies of kinetics of cellular influxes, timing after antigen challenge, and relative comparison with increases in other phenotypes. Any pathogenetic role assigned to a phenotype must also await functional studies, including cytokine generation and secretion and cell-cell interactions in situ.

Adoptive transfer of experimental hypersensitivity pneumonitis in the LEW rat.

The pathogenesis of immune-mediated pulmonary inflammation following inhalation of antigen in sensitized subjects may involve specific cellular or humoral mechanisms, or both. We used adoptive transfer to document cellular mechanisms in an established LEW rat model of acute hypersensitivity pneumonitis. The optimal protocol utilized sensitized spleen cells cultured for 72 h in the presence of antigen and concanavalin A before intraperitoneal injection of 20 x 10(6) cultured cells into sublethally irradiated recipients. Experimental and control rats were subjected 7 d later to twice daily aerosol challenge for 1 or 3 d and examined 12 h after the last challenge. Histopathology was evaluated in one lung, and the other was lavaged and cells evaluated by flow cytometry for T cell phenotypes (CD4, CD8, and RT6, CD45R subsets). Histopathology and cell numbers for most phenotypes were increased in experimental over controls, but proportions were unchanged. Similar results were obtained in animals evaluated at both 24 and 72 h after initial challenge. Results indicate cellular participation in an established LEW rat model of HP. Significant increases in numbers of T cell phenotypes suggest an important role in pathogenesis, but no single phenotype could be incriminated by disproportionate numbers.

Prognostic value of methacholine challenge in patients with respiratory symptoms.

BACKGROUND: The objective was to study the current clinical status of 78 adults with respiratory symptoms, who were referred 3 to 10 years ago for diagnostic methacholine challenge. We tested the hypothesis that methacholine hyperresponsiveness would be associated on follow-up with increased symptoms of chest tightness, dyspnea, wheezing, cough, and more frequent use of selected treatment modalities. METHODS: Current symptoms were evaluated by means of interview questionnaire, and methacholine challenge was repeated during follow-up examination. Comparisons were made between patients who were and those who were not hyperresponsive to methacholine at initial and follow-up challenges by using specific symptoms and calculated symptom and treatment scores. RESULTS: We found that subjects who had positive methacholine challenge results on initial challenge (n = 37) were significantly more likely than those with negative results (n = 41) to have nonexertional chest tightness, wheezing, and dyspnea, but not cough. A high proportion of both groups had current symptoms. Two thirds of the patients continued to have positive (n = 25) or negative (n = 27) methacholine challenge results, and one third had a change in status (n = 26). Significant correlations were also found between follow-up methacholine responsiveness and concurrent symptoms, again with the exception of cough. CONCLUSIONS: Methacholine challenge warrants cautious interpretation in the individual patient as an aid to diagnosis and prognosis in the evaluation of respiratory symptoms, especially cough.

Methods for survey and diagnosis.

This contribution is an overview of current epidemiologic methods used in evaluating organic dust-containing environments for evidence of adverse pulmonary effects. Potential problems include vague diagnostic criteria of "disease" and inadequate tools for definitive diagnosis, as illustrated by asthma and hypersensitivity pneumonitis. The enthusiastic investigator may consider trivial changes indicative of real or potential disease, and add the studied environment to a growing list of menaces of the workplace or of everyday life. Attention to scientific principles is needed lest epidemiology becomes scare-mongering made respectable by sophisticated statistics.

Comparisons of specific and nonspecific bronchoprovocation in subjects with asthma, rhinitis, and healthy subjects.

BACKGROUND: We studied subjects with atopic asthma, atopic rhinitis, and nonatopic healthy subjects to evaluate responsiveness to bronchoprovocation with both methacholine and allergen. METHODS: Subjects with a demonstrable FEV1 PD20 to methacholine or allergen (responders) were further analyzed for putative sensitivity (PD20 FEV1) and reactivity (dose-response slopes) to determine whether any characteristics could distinguish individuals with asthma from other responders. Subjects were recruited without sex restrictions and were between the ages of 18 and 45 years old. They were nonsmokers, had no other medical problems, and were free of upper respiratory infection for at least 6 weeks before challenge. All had a history taken, physical examination, limited laboratory screening, chest radiography, pulmonary function testing, and intradermal skin testing before admission to the study. RESULTS: Although the groups were significantly different in both sensitivity and reactivity to methacholine, responses to allergen bronchoprovocation were sufficiently similar between responders with asthma and those with rhinitis to prevent separation on the basis of either sensitivity or reactivity. The fall in FEV1 at the nadir of the late response, which was greater in the asthma group, was significantly correlated with sensitivity and reactivity of the immediate response to allergen but not to methacholine. Regression analysis demonstrated a stronger association between allergen and methacholine responsiveness in subjects with rhinitis than in subjects with asthma. CONCLUSION: We concluded that (1) nonspecific bronchial hyperresponsiveness fails to explain why patients with allergic asthma have clinical asthma as a result of allergen exposure and patients with allergic rhinitis do not; (2) hyperresponsiveness to allergen does not simply reflect quantitative or qualitative airway nonspecific hyperresponsiveness; and (3) clinical asthma may involve mechanisms difficult to elucidate by laboratory bronchoprovocation techniques.

Experimental hypersensitivity pneumonitis in the rat: histopathology and T-cell subset compartmentalization.

The pathogenesis of hypersensitivity pneumonitis (HP) appears to depend largely on T-cell specificity and interactions with monocytes/macrophages. We studied T-cell subset populations, defined by surface membrane markers with putative functional correlates, present in lung parenchyma and bronchoalveolar spaces of sensitized LEW rats following acute and chronic inhalational challenges with antigen. Our initial hypothesis was that the CD4+ RT6- (TDH) subset, the effector cell of delayed hypersensitivity, would dominate in HP lesions and that CD8+ RT6+ (TS) or CD8+ CD45R+ (TS) subsets, constituting putative suppressor T-cell populations, would dominate in lungs of animals with resolving lesions. We found, however, a heterogeneous population involving all eight of the T-cell subsets that were evaluated. Percentages of the RT6+ phenotype diminished as T cells moved from peripheral blood to lung. Dominant numbers of T cells in acute HP included CD8+ RT6- (TCYT) and CD8+ CD45R- (TCYT) subsets, with putative cytotoxic T-cell activity, in addition to CD4+ RT6- (TDH) cells. We did not demonstrate increases in T suppressor cells as the disease waned.

The antigen-limited nature of microtiter ELISAs requires partial depletion of IgG to permit reliable determination of rabbit serum IgA antibody activity.

The antigen-limiting nature of microtiter ELISAs predicts that antibodies of minor classes may be underestimated when the same specimen contains large amounts of IgG antibodies specific for the same antigen. Such competitive inhibition can be diagnosed from ELISA titration plots. A method is described to eliminate the negative effects of this competition on the detection of IgA antibodies in rabbit serum. The detectability of rabbit serum antibodies to ovalbumin and bovine serum albumin is increased 10-fold by prior treatment of 1:100 dilutions of serum with 1% Cowan I S. aureus. High concns of S. aureus, e.g. 10%, completely deplete serum IgG without loss of IgA. However, concns higher than 1% do not lead to additional improvement in the detectability of IgA antibodies in the systems studied. The method is rapid, inexpensive and shows no non-specific depletion of IgA from either serum or bronchoalveolar lavage fluid.

Uptake of muramyl dipeptide fluorescent congeners by normal rabbit bronchoalveolar lavage cells: a study using flow cytometry.

Muramyl dipeptide (MDP) is the minimal adjuvant-active structure of mycobacterial cell walls and is known to activate monocytes/macrophages, but mechanisms involved with uptake and activation of these cells have not been completely defined. Earlier studies addressing uptake of MDP and the question of receptors have utilized radioligands and murine peritoneal macrophages. We used fluorescent congeners of MDP and flow cytometry to explore kinetics and specificity of uptake by bronchoalveolar cells of normal rabbits. Both washed cells and cell suspensions from which the fluorescent congeners were not washed were used, and incubation was carried out primarily at 4 degrees C. Fluorescence microscopy consistently revealed intracellular but no visible membrane fluorescence of alveolar macrophages. Uptake was dose dependent but was not saturable up to concentration limits of fluoresceinated muramyl tripeptide (MTP-FITC) imposed by the system, and was partially inhibited by excess unlabeled MDP, consistent with specific inhibition. Alveolar macrophages, but not lymphocytes, demonstrated specific uptake at 4 degrees C, with rapid on- and off-times. Uptake was enhanced 7-fold at 37 degrees C. Uptake was greater by larger, more granular macrophages than by smaller, less granular macrophages, but no difference in uptake was found when cells of similar size but different densities were compared. The exact mechanism of the rapid uptake at 4 degrees C is uncertain but appears to be competed for by unlabeled MDP.

Unifying concepts underlying the effects of organic dust exposures.

Defense mechanisms protect the lung very well from inhaled organic dusts. With sufficient exposure to certain dusts, however, susceptible individuals develop hypersensitivity pneumonitis (HP), the organic dust toxic syndrome (ODTS), or asthma. Mucous membrane irritation (MMI) bothers some individuals inhaling grain dust. Allergic asthma is caused by IgE-mediated immunologic responses to allergenic dust contaminants. ODTS can be explained by a nonimmunologic release of interleukin 1 (IL-1) and perhaps other endogenous pyrogens from alveolar macrophages by endotoxin or other ingredients of dusts. The pathogenesis of HP may involve IL-1 release combined with a specific immunological response by effector T-lymphocytes. MMI may be the result of an irritant effect not involving immune responses or mediators.

Guidelines for the clinical evaluation of hypersensitivity pneumonitis. Report of the Subcommittee on Hypersensitivity Pneumonitis.

In general, a history of exposure to "moldy" hay, birds, or other incriminated occupational or environmental inhalants in a patient with clinical and radiologic features consistent with HSP should lead to the demonstration of serum precipitins to the suspected antigen and an established diagnosis, confirmed by avoidance of the agent involved. Occasionally, other diagnostic procedures are required. The diagnosis is often difficult in domestic exposures, such as humidification and air conditioning systems. A careful environmental history is essential, and at times the physician must inspect the patient's environment personally. In most cases, the diagnosis is established if (1) the history and physical findings and pulmonary function tests indicate an interstitial lung disease, (2) the x-ray film is consistent, (3) there is exposure to a recognized cause, and (4) there is antibody to that antigen. In other exceptional circumstances, bronchoalveolar lavage may help. Biopsy is rarely needed. Special environmental studies and identification of new antigens require research facilities. Provocation tests are research procedures, not necessary for the diagnosis, and not needed in contested workmen's compensation adjudications.

Use of segmental airway lavage to obtain relevant mediators from the lungs of asthmatic and control subjects.

Studies have demonstrated that increased amounts of histamine in the airways of asthmatic patients are associated with increased airway reactivity. However, using routine bronchoalveolar lavage (BAL), histamine can be detected in only a portion of asthmatic subjects and a minority of control populations. To obtain relevant mediators from the airways in higher concentrations by avoiding the dilution inherent with a standard BAL, a technique was developed to lavage isolated airway segments of the human lung that employed a double-lumen bronchoscope and a balloon-tipped catheter. Lavage fluid obtained by this method yielded significantly higher concentrations of histamine than that obtained with routine BAL (asthmatic subjects, 2,403 +/- 633 pg/ml vs 188 +/- 42 pg/ml; rhinitis subjects, 533 +/- 187 pg/ml vs 113 +/- 53 pg/ml; normal subjects, 174 +/- 63 pg/ml vs 11 +/- 11 pg/ml). Similar findings were also noted for prostaglandin D2 (PGD2). Segmental airway lavage also resulted in higher lavage fluid concentrations of LTB, than routine BAL. Segmental airway lavage should help in studying the relationship of mast cell degranulation to airways reactivity in both asthmatic and other study populations.

Bronchoalveolar lavage T-cell and Ia antigen quantitation by flow cytometry in acute and chronic experimental hypersensitivity pneumonitis.

Increasing evidence implicates effector T-cells in the pathogenesis of hypersensitivity pneumonitis. We utilized T-cell- and Ia-specific antibodies, flow cytometry, and computer analysis to quantitate T-cell numbers and Ia expression on lung cells of established rabbit models of acute and chronic hypersensitivity pneumonitis (HSP). We used analysis of variance (ANOVA) and Turkey's follow-up to evaluate group differences. In the acute HSP group, increased percentages of T-cells and greater Ia expression were present on bronchoalveolar lavage (BAL) cells at 24 h after inhalational allergen challenge. Increased BAL T-cell numbers were found in the chronic HSP group produced by repeated inhalation of antigen and muramyl dipeptide, compared to "desensitized" animals and control groups. Both chronic HSP and desensitized groups demonstrated increased Ia expression on BAL cells. Pathology scores for individual animals in both acute and chronic protocols correlated significantly (Pearson correlation coefficients) with total numbers of BAL cells, percentages of T-cells, and percentages of Ia-positive cells recovered. Findings are consistent with the hypothesis that cell-mediated hypersensitivity is a central mechanism in the pathogenesis of experimental hypersensitivity pneumonitis.

Synthesis of fluorescent muramyl dipeptide congeners. 2.

Muramyl dipeptide (MDP) analogues were prepared and utilized in the synthesis of new fluorescently labeled MDP derivatives for use as biologic probes. Thus, N alpha-(N-acetylmuramyl)-L-lysyl-D-isoglutamine (Lys-MDP, 4) and N alpha-(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl)-L-lysine [MTP, 5] were synthesized and then reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to yield the fluorescent adducts, DTAF-Lys-MDP (6) and DTAF-MTP (7). The adjuvant activity of the fluorescent MDP derivatives was determined by the ability of the compounds to promote delayed skin test responses in guinea pigs immunized with ovalbumin (OA) and by evaluating the anti-OA activity of these guinea pigs.

Alveolar macrophage immunosuppression is maintained in rabbit models of hypersensitivity pneumonitis.

In established experimental models of hypersensitivity pneumonitis and, perhaps, in exposed, asymptomatic humans, continued aerosol exposure to protein antigen results in waning disease and a state of desensitization. The mechanisms causing such unresponsiveness are not well understood, but a possibility is enhanced immunosuppression by alveolar macrophages or other bronchoalveolar cells. Similarly, a loss of immunosuppressive function could result in the appearance of alveolitis. We therefore compared the ability of bronchoalveolar lavage (BAL) cells to augment or suppress antigen-specific lymphocyte blastogenesis in rabbit models of acute and chronic hypersensitivity pneumonitis, desensitized animals, and control animals. We found that BAL cells from all treatment groups suppressed antigen-specific lymphocyte blastogenesis at BAL:lymph node cell ratios of 1:1 to 1:8. BAL cells from some animals were suppressive at high BAL concentrations and, at lower concentrations, augmented the blastogenic response. Additional studies revealed no significant differences in the ability of mitomycin C-treated BAL cells to suppress or augment autologous lymphocyte blastogenesis at any ratio tested. Low-density, macrophage-enriched BAL cells obtained by Percoll fractionation maintained suppressive function. Addition of indomethacin to cultures only partially abrogated BAL-mediated suppression of antigen-specific blastogenesis. We conclude that the development of alveolitis in this model cannot be attributed to loss, nor can desensitization be explained by augmentation, of alveolar macrophage immunosuppressive function.