Flow cytometry analysis of T lymphocytes in sarcoidosis

To examine the immunologic alterations in patients with sarcoidosis we characterized the cell cycle phase of T lymphocytes that were collected from peripheral blood and from bronchoalveolar lavage fluid. T-cell DNA and RNA contents were measured at the single cell level using flow cytometry after staining with the metachromatic fluorochrome acridine orange. T-enriched lymphocyte suspensions were obtained from peripheral blood and from bronchoalveolar lavage in 17 patients with histologically-proved sarcoidosis (10 patients, stage I and 7 patients, stage II) and in 4 patients with acute extrinsic hypersensitivity pneumonitis (AEHP). The percentages of cells in the S + (GS + M) phase in the peripheral blood of the patients with sarcoidosis did not differ from those of healthy control subjects. With bronchoalveolar lavage, however, elevated numbers of T cells in the S + (G2 + M) phase were found in the patients with AEHP and in those with stage II sarcoidosis when compared to patients with stage I sarcoidosis. The cellular RNA content of T lymphocytes from the peripheral blood showed a typical bimodal distribution without difference between patients and control subjects. Conversely, T lymphocytes obtained by bronchoalveolar lavage from patients with AEHP and sarcoidosis had a homogeneous low RNA content which differed from that of T lymphocytes from the blood from that of in vitro phytohemagglutinin-stimulated lymphocytes. These findings provide a new approach to the study of the mechanisms of local T-cell activation in sarcoidosis.     

Bronchoalveolar lavage and lung histology. Comparative analysis of inflammatory and immunocompetent cells in patients with sarcoidosis and hypersensitivity pneumonitis

To determine whether bronchoalveolar lavage reflects the histologic aspects of the lung histology in patients with sarcoidosis and hypersensitivity pneumonitis, cells recovered from lavage fluid were compared with tissue sections from transbronchial lung biopsies in 33 patients. The evaluation of cellular types and their topographic distribution in situ was determined by using monoclonal antibodies in combination with immunohistochemical techniques. Cell counts in bronchoalveolar lavage and lung biopsies were significantly correlated both in sarcoidosis and hypersensitivity pneumonitis. In fact, the relative proportions of inflammatory and immunocompetent cells recovered from lavage fluid accurately overlapped those observed in lung tissue sections. However, in patients with more pronounced alveolitis, the frequency of macrophages in tissue sections was higher than that observed in the bronchoalveolar lavage, and the degree of lymphocytes in the lavage was higher than that observed in the corresponding biopsy. Specifically, in these patients the lavage underestimated the amount of macrophages in the lung biopsies and overestimated the number of lymphocytes that were present in the lung parenchyma. This was more evident in patients with hypersensitivity pneumonitis, where the intensity of alveolitis was higher than in sarcoidosis. Our data support the idea that, at least in patients with sarcoidosis and hypersensitivity pneumonitis, bronchoalveolar lavage correctly samples the alveolitis. Discrepancies in patients with very high intensity alveolitis could be due to a more pronounced recirculation of lymphocytes from the parenchyma to the alveolar spaces.     

Helper T-lymphocytes in pulmonary sarcoidosis. Functional analysis of a lung T-cell subpopulation in patients with active disease

Pulmonary sarcoidosis is a disease characterized by increased numbers of T-lymphocytes in the alveolar structures, which through the production of lymphokines modulate granuloma formation and polyclonally activate B cells to secrete immunoglobulins. The T-lymphocyte alveolitis is associated with a different expansion of various T-cell subpopulations identified by different monoclonal antibodies. Patients with active disease have increased numbers of helper T cells in the lungs, recognized by the OKT4 monoclonal antibody and decreased numbers of suppressor OKT8-positive lung T cells, whereas patients with inactive disease have increased numbers of OKT8-positive T cells and decreased numbers of OKT4-positive T cells in the lungs. Using the IgG fraction of a monoclonal antibody called 5/9, which reacts in normal subjects with approximately 30% of the OKT4-positive T-lymphocytes, it has been shown that the 5/9-positive T cells appear preferentially expanded in pulmonary sarcoidosis at sites of disease activity. To evaluate the functions of the T-cell subpopulation identified by the 5/9 monoclonal antibody in pulmonary sarcoidosis, we studied the unfractionated T-lymphocytes and the 5/9-positive and the 5/9-negative T-cell fractions in bronchoalveolar lavage of 12 patients with active lung disease. On T-cell suspensions, the spontaneous release of monocyte chemotactic factor and the polyclonal activation of autologous peripheral blood lymphocytes were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiproteases are increased in bronchoalveolar lavage in interstitial lung disease

The present study evaluates different cellular and soluble components in the bronchoalveolar lavage (BAL) from patients with interstitial lung disease. We observed an increased T4/T8 lymphocyte ratio in BAL but not in blood from 24 patients with active pulmonary sarcoidosis compared to sixteen normal individuals and to eleven patients with inactive pulmonary sarcoidosis. Seven patients with hypersensitivity pneumonitis had a normal T4/T8 ratio. In the active sarcoidosis and hypersensitivity pneumonitis groups, alpha 1-Protease Inhibitor (alpha 1 PI) in BAL is significantly higher than in the normal group and a significant correlation between the two antiproteases (alpha 2-macroglobulin and alpha 1 PI) is observed. These data demonstrate that antiprotease levels (alpha 1 PI and alpha 2 M) are increased in the lower respiratory tract of patients with interstitial lung disease and that among cellular and soluble components of BAL, alpha 2 M represents a sensitive marker of the alveolitis.     

Lung injuries and serial bronchoalveolar lavage findings in patients with summer-type hypersensitivity pneumonitis

To evaluate acute lung injuries and their persistence in patients with summer-type hypersensitivity pneumonitis, repeated bronchoalveolar lavage (BAL) and pulmonary function tests were performed. BAL was performed on 36 occasions in 17 patients with hypersensitivity pneumonitis. Nineteen BAL procedures in 16 cases were done during the active phase within the three hospital day and BAL was repeated in 8 cases during inactive phase. Anti-Trichosporon cutaneum antibodies were detected in all of 15 cases examined using the Ouchterlony method and indirect immunofluorescent methods. The number of total BAL cells, lymphocyte and neutrophils were increased in the active phase, and OKT4/OKT8 was quite low (0.39). As the disease became inactive, the number of total BAL cells, lymphocytes and neutrophils decreased. On the other hand OKT4/OKT8 increased quickly. Significantly negative correlations were recognized between the number of BAL lymphocytes and %VC, and lymphocytes and %DLCO4. More improvements in BAL findings, %VC and %DLCO one year after acute episodes were seen in cases that moved house than cases that did not move, and these often relapsed. We concluded that complete clearing of the patient's house or moving out, if necessary, were needed to avoid relapse and persistent lung injuries.     

Bronchoalveolar lavage cells and proteins in patients with the acquired immunodeficiency syndrome. An immunologic analysis

Several components of cellular and humoral immunity were examined in bronchoalveolar lavage fluid and blood of 15 patients with the acquired immunodeficiency syndrome, and the results were compared to data from 25 healthy controls (including 5 asymptomatic homosexual men). Compared with that of controls, bronchoalveolar lavage fluid from patients tended to have more lymphocytes and significantly more neutrophils; a lower OKT4/OKT8 ratio, due to an increase in total OKT8 cells; and normal total OKT4 cell counts, despite a significant decrease in numbers of OKT4 cells in peripheral blood. Patients also had significantly more IgG-releasing cells and higher IgG levels than controls in lavage fluid. These data show that, in the lung lining fluid of patients with the acquired immunodeficiency syndrome, significant alterations in cellular and humoral immunity exist that differ in several important respects from immunity in controls and from corresponding changes in patients' peripheral blood.     

A case of chronic hypersensitivity pneumonitis due to wild pigeons]

A 61-year-old woman was admitted to the Oita Medical University Hospital because of a nonproductive cough and exertional dyspnea. Interstitial changes had been seen on her chest radiograph 5 years previously, but no respiratory symptoms were identified at that time. On admission, chest radiography revealed linear and ground-glass opacities in the middle and lower lung fields. Computed tomography provided evidence of bronchiectasis and micro-honeycombing of the lungs, while lymphocyte and neutrophil counts in the bronchoalveolar lavage fluid were increased. Transbronchial lung biopsy demonstrated alveolitis and Masson's bodies. The patient was not a pigeon breeder, but she could have been exposed to pigeons at her workplace. Indeed, she had specific antibodies against pigeon serum and droppings, and her peripheral lymphocytes showed proliferation in response to pigeon serum. A positive provocation test involving inhalation of pigeon serum confirmed that she had chronic hypersensitivity pneumonitis caused by allergy to pigeons. This is a rare case of chronic hypersensitivity pneumonitis associated with wild pigeons, that progressed to pulmonary fibrosis. Antigen provocation testing proved to be of great value.     

Lymphocyte subpopulations in bronchoalveolar lavage in Sj?gren's syndrome. Evidence for an expansion of cytotoxic/suppressor subset in patients with alveolar neutrophilia

We initiated this study to determine the cellular composition and T-lymphocyte subpopulations of fluid from bronchoalveolar lavage from 15 patients with primary Sj?gren's syndrome (1SS), six patients with secondary Sj?gren's syndrome associated with primary biliary cirrhosis (2SS-PBC), eight patients with secondary Sj?gren's syndrome associated with collagen-vascular diseases (2SS-CVD), and 12 normal subjects. All were nonsmokers who were free of clinical pulmonary symptoms and had normal findings on chest roentgenograms. Lymphocyte subsets were identified by mouse monoclonal antibodies that were specific for T-cells, helper/inducer, and suppressor/cytotoxic (namely, OKT3, OKT4, and OKT8). Patients with 1SS, patients with 2SS-PBC, and patients with 2SS-CVD had a significantly increased percentage of lymphocytes in fluid from bronchoalveolar lavage (respectively, 21.6 +/- 3.7 percent, 24.3 +/- 6.1 percent, and 25.6 +/- 3.9 percent) compared with the normal value of control subjects (9.9 +/- 1.5 percent). In addition, two of the 15 patients with 1SS and five of the eight patients with 2SS-CVD demonstrated an increased percentage of alveolar neutrophils. The predominant T-cell subset in patients with 1SS was T4+, and the mean T4:T8 ratio was normal. The percentage of T4+ cells was increased in patients with 2 SS-PBC, resulting in an increased T4:T8 ratio. In contrast, patients with 2 SS-CVD demonstrated a markedly increased percentage of T8+ cells, reflected by a shift in the T4:T8 ratio which was inverted. Patients with Sj?gren's syndrome and with neutrophilia on bronchoalveolar lavage had a marked expansion of the T8+ lymphocyte subpopulation, where as patients with Sj?gren's syndrome and with pure lymphocytosis on bronchoalveolar lavage showed predominantly T4+ cells. In addition, we found a strong positive correlation between the number of neutrophils and the number of T8+ cells in bronchoalveolar lavage from patients with Sj?gren's syndrome (r = 0.74; p less than 0.05). Until the functional activities of OKT4+ and OKT8+ cells are better defined, the role that these cells play in the pathogenesis of pulmonary disease in Sj?gren's syndrome remains unclear.     

A comparison of albumin and urea as reference markers in bronchoalveolar lavage fluid from patients with interstitial lung disease

Lavage fluids were investigated for 67 subjects in 6 groups: 12 with active sarcoidosis, 8 with inactive sarcoidosis, 17 with pigeon breeder's disease, 10 asymptomatic pigeon breeders, 12 with idiopathic pulmonary fibrosis (IPF) and 8 normal subjects. Albumin and urea per ml of bronchoalveolar lavage fluid (BALF) were determined for each subject together with percentage return of fluid (BAL%). Novel assay systems were employed to measure urea and albumin and these were compared with existing analytical techniques. When compared with the control group, we found that urea per ml of BALF was not statistically different for all other groups, except those with pigeon breeder's disease who had significantly raised levels. For albumin, however, three groups had significantly higher levels than the controls, namely those with active sarcoidosis, pigeon breeder's disease and IPF. BAL% return showed no significant differences for any group when compared with the controls. We conclude that since albumin is significantly raised in most patients with interstitial lung disease it does not represent a suitable marker for the quantitation of reactive proteins in BALF. Urea shows much less variability between groups than does albumin, and hence in the absence of a proven alternative represents the most reliable estimate available of epithelial lining fluid dilution during the lavage procedure, providing dwell time is kept to a minimum.     

IgA and IgG antibody activities of serum and bronchoalveolar fluid from symptomatic and asymptomatic pigeon breeders

Serum IgA and IgG antibody activities against pigeon serum were measured in 16 symptomatic pigeon breeders, 20 asymptomatic pigeon breeders, and 3 normal subjects by radioimmunoassay. The IgA and IgG antibody activities against pigeon antigen of the group of patients with disease was significantly greater than those of patients in the asymptomatic and the control group. The overlap of results for the symptomatic and asymptomatic breeders limits the diagnostic value of these individual IgA or IgG antibody determinations. Bronchoalveolar fluid and serum samples from a smaller group of pigeon breeders who underwent lung lavage were available for studies of antibody activity against pigeon serum. Ten asymptomatic and 6 symptomatic breeders were available for study. Both IgG and IgA antibody activities were detected by radioimmunoassay in serum samples and bronchoalveolar fluid. The IgA antibody activity determined by the radioimmunoassay was higher in the respiratory secretions.     

Determination of T lymphocyte subpopulations in patients with lung cancer. A comparison between lung lavage and peripheral blood by monoclonal antibodies and flow cytometry

In order to determine whether the alterations of immunoregulatory T cells described both in smokers and in patients with lung cancer occur in the deep lung as well as in peripheral blood, we analyzed T lymphocyte subpopulations in bronchoalveolar lavage (BAL) and in the blood of 12 patients with untreated lung cancer and of 8 controls. The immunocompetent cellular population of BAL fluid analyzed by differential cell count of alveolar macrophages, lymphocytes and neutrophils did not show considerable differences in the two groups studied. By contrast, the analysis of BAL T lymphocytes and their subsets showed significant alterations in patients compared with controls: a percentage increase of OKT3+ and OKT8+ lymphocytes and a decrease of the OKT4+/OKT8+ ratio was found in both the involved and uninvolved lung of patients. The immunologic pattern of T lymphocytes in blood did not show significant differences between patients and controls. Our data indicate that alterations in immunoregulatory T cells in lung cancer are more pronounced in BAL fluid obtained from both lungs than in peripheral blood.     

Subpopulations of T cells in lung biopsies from patients with pigeon breeder's disease

Monoclonal antibodies were used to determine surface phenotypes of T cells in tissue obtained by open lung biopsies from patients with chronic hypersensitivity pneumonitis (pigeon breeder's disease). The results indicate that an increased number of suppressor/cytotoxic cells is present in these patients when compared with the number of helper/inducer cells. These findings, which were present within the interstitium, are consistent with those found in bronchoalveolar lavage of patients with this disease. In addition, in two-thirds of the patients there was a greater total number of helper and suppressor cells than the total count for Pan T cells. A possible interpretation of these findings might be the presence of both markers in the same cell.     

Pigeon hypersensitivity pneumonitis: immunohistochemical demonstration of the causative antigen in the lung.

A number of clinicopathological manifestations may define the presence of hypersensitivity pneumonitis. Histological study is used to establish the diagnosis and to differentiate the disease from other respiratory disorders. This case report suggests that immunohistological demonstration of the causative antigen in the lung may be a useful diagnostic approach in cases of pigeon hypersensitivity pneumonitis. A 52 year-old woman was studied. She had a prior history of pigeon exposure, and lived in an area with a high prevalence of tuberculosis. Her clinical presentation, respiratory function tests and imaging studies revealed a predominant interstitial lung disease. The results of antiavian antibodies, bronchoalveolar analysis, and other laboratory parameters were non-diagnostic. A lung biopsy showed a prominent granulomatous reaction with a sarcoid-like appearance in some areas, and an interstitial infiltration constituted by lymphocytes, plasma cells and foamy macrophages. Although the disease manifestations were compatible with hypersensitivity pneumonitis, we decided to study the causal antigen by immunohistochemistry. The use of a polyclonal antibody raised against pigeon serum showed a predominant cytoplasmic immunostaining in multinucleated giant cells and histiocytes from lung granulomas. Other respiratory disorders were reasonably excluded. Previous exposure to a known antigen may support the diagnosis of hypersensitivity pneumonitis. Although the inhalation of organic dusts may be clinically evident, the aetiology is commonly evaluated by different challenge tests or immunological methods. We propose that the study of pigeon antigen by immunohistochemistry may be used as part of the diagnostic approach for hypersensitivity pneumonitis.     

Subpopulations of T cells in lung biopsies from patients with pigeon breeder’s disease

Monoclonal antibodies were used to determine surface phenotypes of T cells in tissue obtained by open lung biopsies from patients with chronic hypersensitivity pneumonitis (pigeon breeder’s disease). The results indicate that an increased number of suppressor/cytotoxic cells is present in these patients when compared with the number of helper/inducer cells. These findings, which were present within the interstitium, are consistent with those found in bronchoalveolar lavage of patients with this disease. In addition, in two-thirds of the patients there was a greater total number of helper and suppressor cells than the total count for Pan T cells. A possible interpretation of these findings might be the presence of both markers in the same cell.     

Functional significance of anti-T-lymphocyte antibodies in sarcoidosis

Pulmonary sarcoidosis is a chronic disorder characterized by the activation of helper/inducer T-cells in the lung without a concomitant increase in suppressor/cytotoxic T-cells. It is known that patients with sarcoidosis have circulating anti-T-cell antibodies, primarily of the IgM class. To evaluate a functional role for these antibodies in enhancing lung helper T-cell processes in pulmonary sarcoidosis, we evaluated serum and lavage fluid of patients with active sarcoidosis for the presence of anti-T-cell antibodies, the T-cell subset specificity of these antibodies, and the possible stimulatory or inhibitory effects of these antibodies on T-cells relevant to the exaggerated helper T-cell processes in sarcoidosis. Indirect immunofluorescence studies demonstrated that sarcoid patients had anti-T-cell antibodies of the IgM type reacting with autologous as well as with nonautologous normal T-cells. IgM recovered in sarcoid lavage fluid also reacted with T-cells, thus demonstrating the autoantibodies at the site of disease. Two-color immunofluorescence and flow cytometry showed that these sarcoid autoantibodies bound to mostly Leu2+ suppressor/cytotoxic T-cells, but also to a small proportion of Leu3+ helper/inducer T-cells. Incubating lymphocytes with sarcoid serum or IgM purified from sarcoid serum did not stimulate T-cell proliferation. Furthermore, when Leu2+ T-cells were stimulated with irradiated allogenic B-cells, increasing concentrations of sarcoid serum had no inhibitory effects on the activation and proliferative response of the Leu2+ T-cells. Likewise, the purified IgM anti-T-cell antibodies had no inhibitory effects on the mitogenic response of Leu2+ T-cells to the anti-T-cell antigen receptor-associated T3 complex antibody OKT3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bronchoalveolar lavage in extrinsic allergic alveolitis

The precise immunopathological mechanism of extrinsic allergic alveolitis explaining the clinical picture as well as the pathological findings is not known. Bronchoalveolar lavage can be a diagnostic help and a method to unravel the pathophysiology of this disease. In the acute stage of extrinsic allergic alveolitis or within 24 h after antigen exposure an increase in the number of neutrophils is seen. After the acute stage, the number of lymphocytes is even higher than in sarcoidosis. In extrinsic allergic alveolitis as well as in sarcoidosis these lymphocytes are mainly T lymphocytes. However, the distribution of OKT 4 and OKT 8 positive lymphocytes was clearly different in both diseases. In sarcoidosis OKT 4 lymphocytes predominate (OKT 4/8 = 7.8) while in extrinsic allergic alveolitis an increase of both OKT 4 and OKT 8 lymphocytes has been found (OKT 4/8 = 1.5). Whether a type III Arthus reaction or a type IV delayed hypersensitivity with an early component is involved, is discussed.     

Immunology of interstitial lung diseases: cellular events taking place in the lung of sarcoidosis, hypersensitivity pneumonitis and HIV infection

This paper summarizes our research and the results obtained on the topic of immunology of interstitial lung disorders. Areas of investigation mainly included sarcoidosis, hypersensitivity pneumonitis (HP), and more recently the pulmonary involvement in acquired immunodeficiency syndrome (AIDS). In sarcoidosis patients two major mechanisms account for the alveolitis, i.e. an in situ cellular proliferation and a cellular redistribution from the peripheral blood to the sites of disease activity, including the lung. These findings involve both lymphocytes (CD4 helper-related cells) and macrophages, and lead to the formation and provide maintenance of sarcoid granuloma. In patients with hypersensitivity pneumonitis the lung infiltrates are characterized by cells bearing suppressor/cytotoxic phenotype. The expansion of cells with these characteristics in the lung of these patients is likely to be related to a local immune response to the antigenic stimulus. In the lung of patients with AIDS we also found a discrete lymphocytic alveolitis bearing the CD8 cytotoxic-related phenotype. The role of cytotoxic events, related to the lymphocytes and macrophages, which are operative in the lung of AIDS patients, is being evaluated. The analysis of cells recovered from the lavage, mainly lymphocytes and macrophages, in terms of surface phenotype, functional in vitro evaluations and molecular analysis, has provided new insights into the pathogenesis of the above quoted interstitial lung disorders.     

A case of pneumonitis due to serrapeptase

A case of pneumonitis due to Serrapeptase was described. A 69-year-old man was treated with Serrapeptase for 16 days because of common cold, then fever, nonproductive cough and dyspnea developed and chest X-ray revealed diffuse fine granular shadows in bilateral lung fields. Once the administration of Serrapeptase was halted, symptoms, chest X-ray abnormalities and laboratory data improved markedly. The fraction of lymphocytes increased in bronchoalveolar lavage fluid and OKT4/T8 decreased. Microscopic examination of transbronchial lung biopsy showed interstitial pneumonia. Both leukocyte migration inhibition test and sensitized hemagglutination test were positive for Serrapeptase. Based on these findings, we diagnosed this case as Serrapeptase-induced pneumonitis.     

Bird fancier's lung which developed in a pigeon breeder presenting organizing pneumonia

Bird fancier's lung (BFL) is one of the most common types of hypersensitivity pneumonitis. We report a rare case of acute-on-chronic bird fancier's lung that developed in a pigeon breeder and presented subpleural curvilinear shadow and ground glass opacity on high-resolution computed tomography (HRCT) of the chest. The results of surgical lung biopsy showed mainly intraalveolar organization and alveolitis in addition to the pattern of usual interstitial pneumonia with centrilobular fibrosis. Examination of bronchoalveolar lavage (BAL) fluid revealed an increase in lymphocytes. The results of immunoglobulin (Ig) G and IgA antibodies against pigeon dropping extracts were positive in sera and BAL fluid. Consequently, the patient was diagnosed as having BFL. Avoidance of pigeons and corticosteroid therapy led to rapid improvement.     

Local pulmonary immunity in pigeon breeder's disease. A case study

We studied pulmonary and systemic aspects of the immune response in a patient with pigeon breeder's disease before and after an inhalation challenge with pigeon serum. Macrophage migration inhibition was induced with bronchoalveolar wash cells exposed to both pigeon serum and pigeon dropping extract before but not after challenge. Peripheral blood lymphocytes exposed to these antigens did not induce inhibition of guinea pig peritoneal macrophage migration before challenge. However, after challenge peripheral blood lymphocytes did cause macrophage migration inhibition when exposed to pigeon serum. Both systemic and bronchoalveolar lymphocytes proliferated when exposed to these pigeon antigens in vitro. Our patient represents the first reported case of lymphokine production by pulmonary as well as systemic lymphocytes in hypersensitivity pneumonitis.