BACKGROUND--Sarcoidosis is a disease characterised by clinical "anergy" to delayed type hypersensitivity antigens and the formation of non-caseating granulomas, which frequently manifests in the lungs as a T lymphocyte/mononuclear cell alveolitis. Although there is an increased proportion of T cells in bronchoalveolar lavage (BAL) samples from these patients, and these T cells often show evidence of activation and spontaneous secretion of cytokines such as interleukin 2 (IL-2) and interferon gamma (IFN gamma)--a pattern similar to delayed type hypersensitivity reactions--it is unclear whether both cytokines are produced by the majority of T cells derived from the lungs of patients with sarcoidosis or whether unique subpopulations of T cells produce each cytokine. In this study the properties of T cells cloned from BAL fluid samples of patients with sarcoidosis have been analysed. METHODS--T cells were cloned by limiting dilution using IL-2, phytohaemagglutinin, and irradiated feeder cells. Cloning efficiencies were compared and phytohaemagglutinin induced clonal production of IL-2, IFN gamma, and IL-4 was determined by bioassay (IL-2 and IFN gamma) or ELISA (IL-4). RESULTS--T cells derived from the BAL fluid of patients with sarcoidosis cloned less efficiently than those from blood of the same individuals. Lung derived clones (CD4+ or CD8+) produced IFN gamma more frequently and to a higher titre than blood derived clones, whereas IL-2 production by CD4+ clones derived from BAL fluid was less than that from blood derived clones. Interestingly, IL-4 production by clones from both sites was similar. Analysis of the co-production of IL-2, IFN gamma, and IL-4 by these BAL fluid clones did not demonstrate a predominant "Th1"-like population which has been suggested to underlie delayed type hypersensitivity reactions. CONCLUSIONS--The reduced cloning efficiency of T cells from the lung compared with the blood in sarcoidosis is consistent with, although probably more pronounced than, previous observations in normal lungs and shows that T cell hyporesponsiveness is not overcome in the lungs of patients with sarcoidosis. Furthermore, major differences exist between the cytokine producing potential of T cells derived from the lung and the blood in sarcoidosis, and these parallel the differences in the properties of blood and lung T cells seen in healthy individuals.

γδ T cells are capable of mediating non-major histocompatibility complex (MHC) restricted lysis of a variety of tumour cell lines. The mechanism of this lysis and its significance in tumour immunity are not clear. We have used a panel of five malignant mesothelioma (MM) cell lines, as well as standard tumour targets K562 and Daudi, to investigate some of the factors which could be involved in non-MHC restricted cytotoxicity mediated by γδ T cells. Individual MM ceil lines, representing a panel of lines derived from a single cell type, varied in their susceptibility to lysis by γδ T cell clones. Individual γδ T cell clones also showed unique cytotoxic profiles, and differed in their cytotoxic potential. T cell receptor (TCR) γδ gene usage correlated with the ability of clones to lyse Daudi or K562; clones lysing Daudi expressing Vγ9 and clones lysing K562 expressing VγI subgroup genes. No strict correlation between Vγ and Vδ gene usage and MM reactivity was, however, demonstrable. There was also no correlation between yδ T cell lysis of MM cell lines and the capacity of γδ T cells to produce interferon-γ, tumour necrosis factor-α, interleukin-2 or interleukin-4, nor with their expression of CD8.

gamma delta T cells are capable of mediating non-major histocompatibility complex (MHC) restricted lysis of a variety of tumour cell lines. The mechanism of this lysis and its significance in tumour immunity are not clear. We have used a panel of five malignant mesothelioma (MM) cell lines, as well as standard tumour targets K562 and Daudi, to investigate some of the factors which could be involved in non-MHC restricted cytotoxicity mediated by gamma delta T cells. Individual MM cell lines, representing a panel of lines derived from a single cell type, varied in their susceptibility to lysis by gamma delta T cell clones. Individual gamma delta T cell clones also showed unique cytotoxic profiles, and differed in their cytotoxic potential. T cell receptor (TCR) V gamma gene usage correlated with the ability of clones to lyse Daudi or K562; clones lysing Daudi expressing V gamma 9 and clones lysing K562 expressing V gamma I subgroup genes. No strict correlation between V gamma and V delta gene usage and MM reactivity was, however, demonstrable. There was also no correlation between gamma delta T cell lysis of MM cell lines and the capacity of gamma delta T cells to produce interferon-gamma, tumour necrosis factor-alpha, interleukin-2 or interleukin-4, nor with their expression of CD8.

The characteristics of the T-cell population in the healthy human lung have been investigated by analysing the properties of T-cell clones derived from bronchoalveolar lavage (BAL) samples and comparing them with T cells cloned from the blood of the same individuals. The proportions of CD4+ and CD8+ T cells in the starting populations from BAL and blood were similar although only 14% of BAL T cells were CD45RA+ compared to 70% of blood T cells. The precursor frequency of T-cell clones derived from BAL was less than from blood. The cytokine profiles [after phytohaemagglutinin (PHA) stimulation] of the clones derived from both sources were markedly different and these differences lay in the CD4+ population. BAL-derived CD4+ clones produced interferon-gamma (IFN-gamma) more frequently than did those from blood while blood-derived clones were more likely to produce interleukin-2 (IL-2) than those from BAL. IL-4 was produced by the majority of BAL- or blood-derived clones (93% and 88% respectively) either along with IFN-gamma (BAL) or IL-2 (blood). The cytokine profiles of BAL-derived T-cell clones are consistent with those derived from lung interstitium and suggest that the BAL T-cell populations reflect those in the lung wall. Whether the unique properties of lung T cells are acquired after leaving the blood or whether there is selective entry of T-cell subpopulations into the lung remains to be determined.

Recent in vitro studies suggest that IgE production in adults is co-ordinately regulated by negative signals from gamma IFN-producing CD4+ T-helper-1 (TH-1) and positive signals from IL-4 producing (TH-2) T-cells. Additionally, seroepidemiological evidence has pinpointed infancy as the period of maximum lifetime risk for T-cell sensitization to ubiquitous environmental antigens. The present study sought to elucidate the relationship between these observations, by examination of CD4+ T-cell function in normal children and those genetically at 'high risk' for atopy, spanning the age range (up to 4 years) in which IgE responses to environmental allergens is typically manifest. Immunocompetent T-cell precursor frequencies (determined by cloning at limiting dilution) were markedly reduced in 'high risk' children relative to normals (0.53 +/- 0.29 vs 0.26 +/- 0.19; P = 0.0025). Consistent with reports from other laboratories employing bulk T-cell culture techniques, the gamma IFN producing capacity of CD4+ T-cell clones from both groups of children were markedly reduced relative to adults, and was lowest in the high risk group (P < 0.02). IL-4 production by CD4+ T-cell clones from the normal children was within the adult range, but again was significantly lower in the high risk group (P < 0.00005). This indicates that initial immune responses to environmental allergens in early childhood occur against a background of maturational 'deficiency' in CD4+ T-cell function, and suggests the possibility that variations in the rate of postnatal maturation of T-cell competence may be a contributing factor in the development of differing patterns of immunological responsiveness to environmental allergens.

Patients with fibrosing alveolitis have active inflammation within their lung interstitium. Previous studies have focused on the humoral (immune complex) driven processes. In this study increased pulmonary gamma interferon production has been evaluated. Bronchoalveolar lavage cells were obtained from 40 patients with fibrosing alveolitis, 22 with cryptogenic fibrosing alveolitis, and 18 with connective tissue disease associated (CTD) fibrosing alveolitis. Increased gamma interferon production was seen in 12 (30%) patients and was similar in the two study groups. Up to 512 units/10(6) cells were released over 24 hours, showing that the amounts of gamma interferon released could be as large as those seen in other pulmonary diseases associated with active cellular immune processes, such as sarcoidosis. Spontaneous gamma interferon production was related to increased serum concentrations of IgG and IgM but not to serum IgA, antinuclear antibody, or rheumatoid factor titres. There was no relation between gamma interferon production and pulmonary uptake of gallium-67 citrate. The ratio of helper-inducer (Leu-3) to suppressor-cytotoxic (Leu-2) cells in bronchoalveolar lavage fluid was similar in the two study groups and was similar in patients whose cells produced gamma interferon and those whose cells did not. These data suggest that gamma interferon is released in the lungs of a proportion of individuals with cryptogenic fibrosing alveolitis and CTD-fibrosing alveolitis, suggesting a role for this cytokine in mediating these diseases.

Neutrophils accumulate in the alveoli of asbestos-exposed individuals. In determining whether asbestos fibers induce the release of neutrophil chemotactic factor (NCF) from human alveolar macrophages, alveolar macrophages (10(6) cell/mL) obtained by bronchoalveolar lavage from six non-asbestos-exposed control subjects were exposed to crocidolite (0.1 and 1 mg/mL), chrysotile (1 mg/mL), or medium alone for 4 h, and NCF activity was measured in the supernatants in a 48-well microchemotaxis chamber with polycarbonate membrane filters (pore size, 3 microns) and purified human neutrophils. Alveolar macrophages in medium alone released negligible amounts of NCF (4 +/- 1 neutrophils per high-power field [N/HPF]). When macrophages were exposed to crocidolite (0.1 and 1 mg/mL), significant NCF release occurred (43 +/- 9 and 105 +/- 32 N/HPF, respectively; p less than 0.01 for each amount compare to alveolar macrophages cultured in medium alone). Chrysotile (1 mg/mL) induced similar NCF release (96 +/- 14 N/HPF; p less than 0.01 compared to unstimulated alveolar macrophages). Partial characterization of the NCF by Sephadex G-25 fine gel filtration demonstrated a molecular size of less than 1,000 daltons. These results show that human alveolar macrophages release NCF after exposure to asbestos. Release of NCF by alveolar macrophages in asbestos-exposed individuals may play a central role in the pathogenesis of asbestosis.

Gallium lung scanning is widely used to evaluate pulmonary inflammation in patients with interstitial lung disease but has not previously been reported in crocidolite-exposed workers. In order to characterize the pulmonary inflammation caused by crocidolite inhalation, GLS and BAL findings were related to chest x-ray film changes graded according to the ILO classification of roentgenograms of pneumoconioses. In individuals with roentgenographic evidence of asbestosis (CXR greater than or equal to 1/0, n = 15), 13 had a positive GLS and 13 had an abnormal BAL. In asbestos-exposed individuals with equivocal chest x-ray film changes (CXR 0/1, n = 12), six had a positive GLS and six had BAL changes (both GLS and BAL abnormal in three). In individuals with a normal chest x-ray film (CXR 0/0 n = 8), two had a positive GLS and two BAL changes (both abnormal in 1). These data demonstrate that most subjects with crocidolite-induced asbestosis have an abnormal GLS and BAL. In addition, many individuals with asbestos exposure and equivocal or no chest x-ray film changes have an abnormal GLS and/or BAL, suggesting the presence of active subclinical pulmonary inflammation in these individuals.

Bronchoalveolar lavage (BAL) cell counts are used to assess ‘alveolitis’ in patients with interstitial lung diseases (ILD) but inflammatory cells from airways can contribute to the differential cell count. To determine what BAL volume samples airway cells in patients with ILD we measured the proportion of bronchial epithelial cells (BECs) in four successive 25 ml aliquots in a single lung subsegment in 23 patients with ILD (cryptogenic fibrosing alveolitis (CFA) four, rheumatoid lung (RL) three, asbestosis (ASB) 11, sarcoidosis (SARC) five). Cells recovered from the first two 25 ml lavages exhibited higher proportions of BECs (15±14% and 9±2% respectively) than those from the rermaining two aliquots (3±1%, 3±1%, each P<0.01), suggesting that the first 50 ml BAL preferentially sampled airway cells compared to the second 50 ml BAL. To evaluate airway and alveolar inflammatory cell proportions in ILD we performed two separate 50 ml BALs (samples I and II) in a single subsegment in 38 patients with ILD (CFA seven, RL five, ASB 19, SARC seven) and measured the proportions of recovered cells in each sample separately and combined. Seven control individuals were also studied. Sample I contained 1–67% (mean 26±3%) of the total recovered cells. Neutrophil (PMN) proportions were higher in sample I compared to sample II in CFA (20±6 vs 8±2%), RL (30±9 vs 8±2%) and ASB (12±2 vs 7±1%), P<0.05 for each, but were similar in samples I and II in patients with SARC (3±1 vs 2±1%) and controls (2±1 vs 2±1%). In combined samples (I+II), absolute PMN proportions were up to 8% higher than in sample II alone whereas absolute lymphocyte proportions were up to 8% less than in sample II alone. These data suggest that separate processing of the fluid recovered from the first 50 ml BAL in ILD patients provides information on the location of inflammatory cells and improves the accuracy of BAL cell counts.

In order to determine if disordered cellular immune processes are present in the lungs of persons with asbestosis, we performed bronchoalveolar lavage (BAL) on 26 patients with either crocidolite- or chrysotile-induced pulmonary asbestosis and measured the spontaneous release of gamma interferon (IFN gamma), a marker of increased cellular immune activity. For comparison, 18 control subjects and 7 patients with active pulmonary sarcoidosis were also studied. Recovered BAL cells were cultured for 24 h (5 x 10(6)/ml), and the supernatant was assayed for interferon by determining inhibition of cytopathic effect on encephalomyocarditis virus-induced lysis of WISH cells and characterized by monoclonal anti-IFN gamma antibody inhibition. Nine (35%) patients with asbestosis released increased amounts of IFN gamma, up to 320 units/ml, the levels seen in the sarcoidosis patients. All control subjects released less than or equal to 10 units/ml. All interferon released was IFN gamma. In asbestosis patients, IFN gamma production was not related to a history of cigarette smoking, there was no significant difference in the ratio of helper/inducer (Leu-3) to suppressor/cytotoxic (Leu-2) cells in IFN gamma producers compared to non-IFN gamma producers (p greater than 0.05), and IFN gamma production correlated significantly with serum IgG levels (p less than 0.001) but not with the levels of IgM, IgA, antinuclear factor, or rheumatoid factor. These data suggest that active cellular immune processes are present in the lungs of a proportion of patients with asbestosis.

Alveolar neutrophil accumulation occurs in asbestosis. To evaluate a possible role for release of neutrophil chemotactic factor (NCF) in the pathogenesis of asbestosis, spontaneous NCF release from alveolar macrophages obtained by bronchoalveolar lavage (BAL) in eight individuals with asbestosis, 13 asbestos-exposed individuals without asbestosis, and five control subjects has been studied. Alveolar macrophages were incubated in medium (four hours; 37 degrees C), and neutrophil responses to the supernatants were assayed in a microchemotaxis chamber. Alveolar macrophages from subjects with asbestosis released more NCF (97 +/- 19 neutrophils per high-power field [N/HPF]) than controls (3 +/- 1 N/HPF; p less than 0.01). Alveolar macrophages from individuals with asbestos exposure and increased BAL neutrophil proportions (n = 7) released more NCF (93 +/- 24 N/HPF) than individuals with asbestos exposure and normal BAL neutrophil proportions (n = 6; 11 +/- 6 N/HPF; p less than 0.02). The results show that spontaneous NCF release occurs in asbestosis and that NCF release is associated with neutrophil alveolitis in asbestos-exposed individuals without asbestosis, suggesting a pathogenic role for NCF in mediating this neutrophil alveolitis. The results of the study also suggest that the presence of crackles is a better predictor of the presence of neutrophil alveolitis than is an abnormal chest x-ray film.

In order to compare the frequency of elevated lymphocyte proportions and elevated Leu3/Leu2 (helper/inducer to suppressor/cytotoxic) ratios in sarcoidosis with those in clinically similar interstitial lung diseases (ILDs), bronchoalveolar lavage (BAL) lymphocyte proportions and Leu3/Leu2 ratios were determined for 20 patients with sarcoidosis and 30 patients with other (non-sarcoidosis) ILDs [cryptogenic fibrosing alveolitis (seven), lung connective tissue disease (seven), extrinsic allergic alveolitis (four), lymphoproliferative disease (two), and asbestosis (ten)], as well as in eight control subjects. BAL lymphocyte proportions were elevated above control values in 19 (95%) patients with pulmonary sarcoidosis (mean value 36 +/- 3%) but also in 12 (39%) patients with non-sarcoidosis ILDs (mean value 17 +/- 4%). Leu3/Leu2 ratios were over 2.5 in 15 (75%) of the sarcoidosis patients (mean ratio 4.4 +/- 1). In all control subjects, however, and in all but one of the non-sarcoidosis groups, Leu3/Leu2 ratios were below 2.5 (p less than 0.001 for each compared to sarcoidosis patients). Helper/suppressor ratios over 2.5 are found in the majority of our sarcoidosis patients and, unlike raised lymphocyte proportions alone, are not commonly seen in other ILDs, suggesting that evaluation of BAL helper/suppressor cell ratios may be of value in the investigation of patients with ILD.

Bronchoalveolar lavage (BAL) findings in 27 individuals with crocidolite- or chrysotile-induced asbestosis were compared to BAL findings in 29 unexposed control subjects. Alveolitis, defined as an increase in the proportions and/or absolute numbers of inflammatory cells present in BAL fluid compared to values in control subjects, was present in 26 (96 percent) subjects with asbestosis. Most exhibited a neutrophil-eosinophil alveolitis, with neutrophil proportions increased to 7.4 +/- 0.7 percent and eosinophil proportions increased to 2.2 +/- 0.4 percent, compared to 2 +/- 0.5 percent and 0.4 +/- 0.01 percent, respectively, in control subjects (p less than 0.01 for both neutrophils and eosinophils). An increase in the total number of neutrophils and eosinophils per ml of lavage fluid was also seen (neutrophils 23 +/- 5 and eosinophils 13 +/- 4 per ml; p less than 0.05 compared to control subjects). Severity of the alveolitis, defined by the neutrophil or eosinophil proportions, was independent of a history of exposure to cigarette smoke. The pattern and severity of alveolitis in crocidolite- and chrysotile-induced asbestosis were similar. There was a significant correlation between duration of exposure to asbestos and neutrophil proportions (p less than 0.01). No significant difference in the severity of the alveolitis was observed between individuals with radiologic and physiologic evidence of asbestosis compared to those with asbestos exposure and crackles alone, suggesting that, in asbestosis as in other chronic interstitial lung diseases, radiologic and physiologic parameters do not reflect the severity of the alveolitis. This study demonstrates that a neutrophil-eosinophil alveolitis is present in individuals with crocidolite- and chrysotile-induced asbestosis, that this alveolitis is independent of cigarette smoking, and that the severity of the BAL changes is not reflected in radiologic and physiologic changes.