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We have studied chemotactic factor-induced 'complement receptor enhancement' to determine whether changes in receptor expression and complement-dependent cytotoxicity were associated with alterations in cell density. Ficoll-Paque separated normal human neutrophils (>90%), when further fractionated on discontinuous metrizamide (MTZ)gradients(18, 19, 20, 21, 22. 23% MTZ). consistently gave two major bands at the 20/21% and 21/22% interfaces. Incubation with the synthetic chemotactic peptide, N- formyl-methionyl-leucyl-phenylalanine (fMLP (10-8 M)), converted virtually all neutrophils to low density cells sedimenting on MTZ at the 18/19% and 19/20% interfaces. There was a time-dependent change of density after fMLP-stimulation which was maximal at 30 min, with cells reverting towards normal density by 60 min. Control unstimulated cells did not alter their density at any of the time points examined. Activated, low density neutrophils had increased expression of CR1 and CR3 (as shown by flow cytometry and the uptake of -125-F(ab')2 monoclonal anti-CR1 antibody (E11)). These cells also showed enhanced cytotoxic capacity in vitro for helminthic targets (schistosomula of Schistosoma mansoni) opsonized with autologous complement. There were highly significant correlations between cell density and anti-CRI uptake (P<0.001), and between schistosomular killing and change in density (P<0.001). Increased CR1 expression also correlated with enhanced helminthieidal capacity of neutrophils (P<0.001). Complement dependent cytotoxicity was partially reduced after treatment of cells with anti-human CR1 and :or CR3 antibodies, but only in the presence of a second antibody. These findings indicate that chemotactic factor-induced complement receptor enhancement of human neutrophils is associated with a decrease in cell density and increased complement-dependent cytotoxicity (CTX). [ABSTRACT FROM AUTHOR]

We have compared the rosette technique (using C3b-eoatcd red cells) with the uptake of a 125I F(ab')2 anti-complement receptor type I (CR1) monoclonal antibody (Ell) for studying the phenomenon of chemotactic factor (fMLP)-induced complement receptor enhancement (CRE) on human neutrophils. With both methods the dose responses of fMLP were similar with maximal C RE being observed al 10-7 moles 1-1. Conversely, the time course of CRE and the effects of disodium cromoglycate (DSCG) on inhibition of enhancement were discrepant. Maximal CRE with the rosette method was observed al 30 min, whereas increased uptake of anti-CR1 had still not reached a plateau after 1 h. DSCG inhibited fMLP-induced CRE, as assessed by the rosette technique, whereas this agent had no effect on increased binding of radio-labelled anti-CR1 antibody. These results suggest that in CRE the increased adherence of C3b-coated red cells to fMLP-stimulated neutrophils, as measured by the rosette technique, is not dependent exclusively on the increased numbers of CR1. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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-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.

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.

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.

Cell-mediated hypersensitivity has been increasingly implicated in immunologic diseases of the lung, including hypersensitivity pneumonitis (HSP) (extrinsic allergic alveolitis). We used a T cell-specific monoclonal antibody (L11/135) to localize T cells in the parenchyma and bronchus-associated lymphoid tissue of ethanol-fixed, paraffin-embedded lung sections in rabbit models of experimental HSP to define further their possible role in pathogenesis. T cells appeared within 4 hours in early lesions of rabbit models of acute HSP and heavily infiltrated alveolitis lesions at 8 and 24 hours after aerosol challenge. T cells were also present in lesions of rabbit models with chronic alveolitis and occurred peripherally in granulomas. Variable aggregate and follicular forms of bronchus-associated lymphoid tissue rich in T cells occurred in both experimental and control animals. Our findings document early and continuing presence of T cells in lesions in rabbit models of experimental HSP.

One of the diagnostic possibilities to consider when a patient presents with cough, fever, dyspnea, or pulmonary infiltrates is hypersensitivity pneumonitis. Some of the problems encountered in diagnosis of diffuse lung disease are illustrated in two case reports. In one of the cases, interstitial pneumonitis of insidious onset was attributed to inhalation of thermophilic organisms in moldy silage. In the other, the outstanding pathologic feature was bronchiolitis obliterans, and circumstantial evidence pointed to a home humidifier as the source of the problem.

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.

A localized Arthus reaction was produced in the lung of sensitized rabbits by delivery of antigen into a lower lobe bronchus using a method of selective bronchial catheterization under fluoroscopy. The rabbits were sensitized with bovine immunoglobulin G (B-IgG) in incomplete Freund's adjuvant (IFA) to produce precipitating antibody without classic delayed hypersensitivity. Pulmonary histopathology was studied at intervals following antigen challenge, using light and immunofluorescent microscopy. Gross lesions peripheral to the lower lobe bronchus receiving antigen were found within 12 hr. Subsequent necrosis resulted in a dense scar by 6 wk. Microscopically, early lesions were typified by localized bronchitis, bronchiolitis, alveolitis, and vasculitis with exuberant exudates containing predominantly polymorphonuclear leukocytes. Extensive focal necrosis was present by 72 hr. Immunofluorescent studies revealed the presence of B-IgG, rabbit IgG, and complement (C3) in and around bronchi, bronchioles, alveoli, and vessels. No granulomatous lesions were found, and proliferation of alveolar lining cells was not detected in these studies. Thus, the lung can participate in an acute Arthus reaction following local antigen challenge in systemically sensitized animals. The pathology more closely resembles a necrotizing bacterial pneumonia than an interstitial or hypersensitivity pneumonitis under the conditions of this experimental system. Implications for human disease are speculative.

Asthma has been recognized to consist of hyperresponsive airways and cellular inflammation. Allergen bronchoprovocation (BPC) may define the early (EAR) and late-phase asthmatic response (LAR). The LAR has now been associated with increased nonspecific airway hyperresponsiveness and cellular inflammation consisting of neutrophils and eosinophils. We used BPC to demonstrate EAR and LAR in 12 subjects with seasonal allergic asthma. One normal subject and one subject with asthma who had been treated with allergy immunotherapy were challenged but did not respond. Plasma was sampled at frequent intervals during these aeroallergen challenges and assayed for eosinophil chemotactic activity (ECA) and neutrophil chemotactic activity (NCA). Of the 12 subjects with asthma who were challenged, nine had dual responses (both EAR and LAR), and three subjects demonstrated only an LAR. Those subjects who had dual airway responses had biphasic rises in both ECA (early = 267 +/- 28%; late = 286 +/- 28%) and NCA (early = 279 +/- 24%; late = 215 +/- 15%) in their plasma, whereas those subjects who demonstrated only an LAR had only a late rise in ECA (218 +/- 61%) and NCA (188 +/- 31%). The two individuals who did not respond to aeroallergen challenge demonstrated no change in their plasma chemotactic activity toward either eosinophils or neutrophils. Those individuals with the most severe LAR (greater than or equal to 1,000 mm2) had combined ECA plus NCA peak values of greater than 500%.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit models of chronic experimental hypersensitivity pneumonitis and desensitization were used to evaluate the effects of systemic cyclosporine. When administered 12 to 18 h before each inhalational challenge with aerosolized antigen and the adjuvant muramyl dipeptide, cyclosporine suppressed the development of disease as well as the anamnestic antibody response, particularly in bronchoalveolar lavage fluids. When administered at the time of sensitization only, cyclosporine suppressed the primary antibody response but not the anamnestic antibody response or the disease. Antigen- and mitogen-induced blastogenesis was inhibited by cyclosporine in vitro, but antigen-specific blastogenesis was not abrogated by cyclosporine previously administered in vivo. These results indicate that cyclosporine caused profound immunomodulation in this model, which can be at least partially explained by transient suppressive effects on T cells, particularly the helper/inducer and delayed hypersensitivity subset(s).

Chronic asthma and late asthmatic responses (LAR) are associated with local inflammation which might be expected to produce airflow obstruction in small airways and to increase nonspecific airway reactivity. In contrast, early asthmatic responses (EAR) are primarily bronchospastic and probably involve more central airways. We challenged 17 nonsmoking, mildly asthmatic atopic subjects with allergen bronchoprovocation and measured changes in spirometry (FEV1) over the next 24 hours. Each subject also performed a helium-oxygen (He-O2) flow-volume loop before challenge (baseline), during the EAR, and six hours and 24 hours after challenge to measure the effect of gas density on flow rates at midvital capacity. Twelve subjects had both an EAR and a LAR; five subjects had only a LAR. Of these 17 subjects, 15 were initially density dependent, while only two were density independent. During the EAR, 13 percent of the density dependent population had significant decreases in delta Vmax 50 percent; 47 percent had significant decreases during LAR. The He-O2 flow data analyzed at specified time points after challenge revealed significant decreases in the mean delta Vmax 50 percent at six hours in those who had only a LAR (p less than 0.01). In those who had a dual airway response, density dependence increased during the EAR, but decreased at six and significantly at 24 hours (p less than 0.05) postchallenge. There was a strong trend for the severity of the LAR (measured by changes in FEV1) to be directly related to the total decrease in delta Vmax 50 percent during the LAR. We conclude that late asthmatic responses occur frequently after a single antigenic bronchial challenge and can be associated with persistent symptoms. The LAR were often associated with a decrease in density dependence of maximal expiratory airflow, and therefore, may involve small airways.

The effects on the lungs of chronic aerosol and intravenous antigen challenges in preimmunized and control rabbits were studied. Soluble and particulate antigens included ovalbumin, keyhole limpet hemocyanin, antigen-adsorbed latex particles, glutaraldehyde cross-linked ovalbumin, and killed bacille Calmette-Guérin (BCG). Despite the development of acute alveolitis in sensitized animals, chronic aerosol challenge with both soluble and particulate antigens failed to produce chronic interstitial lung disease. Chronic intravenous challenge with killed BCG, but not other particulate antigens, resulted in a progressive interstitial pneumonitis, with evidence of fibrogenesis in animals that had been presensitized to tuberculin by toepad injection of complete Freund's adjuvant. Adaptive alveolar clearance mechanisms thus appear to protect rabbits from chronically inhaled antigen. Pulmonary circulatory clearance of BCG, however, results in an interstitial pneumonitis that is dependent on previous sensitization.

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.

Protamine is used widely to reverse the anticoagulant effects of heparin and to delay the absorption of insulin. Although adverse reactions to protamine are reported infrequently and are usually mild, we recently observed the first fatal case of type I anaphylaxis resulting from protamine. This patient had previously been sensitized to protamine during cardiac catheterization and had high levels of protamine-specific immunoglobulin E in the serum. In a prospective study, we found that 10 of 19 diabetic patients (53%) who had received insulin containing insulin also had high levels of antiprotamine immunoglobulin E. In contrast, none of 27 nondiabetic healthy normal controls or 10 diabetics who had never received protamine or protamine-containing insulin had levels of antiprotamine immunoglobulin E over background. This study underscores the risks of routinely administering protamine to susceptible individuals and the need for alternative therapies.

Intravenous transfusions of washed allogeneic or autologous leukocytes in rabbits resulted in lesions of pulmonary periarteritis 48 hours later. Intact leukocytes were required. Systemic anaphylaxis, generalized Shwartzman reaction, alternate pathway complement activation and inert particle microembolism failed to produce identical lesions. Leukocytes tagged with radioactive chromium were found within arterial thromboses with proximal vasculitis. Generation or release of inflammatory factors plus thromboembolism would explain the pathogenesis of the lesions described. Specific mechanisms may be quite complex. Similar lesions have not been described in studies of pulmonary leukocyte entrapment or experimental microembolism of the lung. This model may be useful for studying pathogenetic mechanisms in pulmonary vasculitis and may have clinical implications.