Activated lung lymphocytes in hypersensitivity pneumonitis

T-lymphocyte activation was investigated in peripheral blood and bronchoalveolar lavage (BAL) of four patients with hypersensitivity pneumonitis. The study was performed by flow cytometry with the use of immunofluorescence labeling with monoclonal antibodies to lymphocyte differentiation or activation antigens. Simultaneous measurement of DNA and RNA content by acridine orange staining was used for cell-cycle analysis. The various cell types were identified by their light-scattering properties. T cell activation was demonstrated in the BAL of all patients by the presence of T cells (OKT3 positive) bearing class II histocompatibility antigen (HLA-DR) and activated T cell markers (MLR 1-3). Lymphocyte proliferation was evidenced in BAL but not in blood of patients by an increased percentage of cells in S + (G2 + M) phases. In addition, T-lymphocyte subsets analysis revealed no abnormalities in the blood and no major imbalance in the BAL despite a slightly increased OKT4/OKT8 ratio. The finding of T cell activation and lymphocyte proliferation in hypersensitivity pneumonitis alveolitis is consistent with the contribution of a local type IV immune reaction to the pathogenesis of this disease.     

Pigeon-serum-induced hypersensitivity pneumonitis in the dog

Pigeon serum (PS) is one of the most common causes of hypersensitivity pneumonitis (HP). PS-induced HP was examined in a dog model. The dogs ( n =6) were immunized by i. m. injections of PS, followed by insufflation with aerosolized PS, while all control dogs ( n =3) received saline only. All animals insufflated with PS developed tachypnea 2–4 h after PS inhalation. After PS insufflation, a significant decrease in arterial oxygen tension (PaO₂) was detected in sensitized dogs. No change in PaO² was detected in sensitized dogs after saline or in the controls after PS insufflation. In intradermal skin tests with PS antigen, a positive skin reaction was found in 3/6 dogs in 30 min, and in 5/6 dogs in 6 and 48 h after the PS injections. Sensitized dogs showed a significant increase in PS-specific IgG in serum and lavage fluid (LF). In LF of sensitized dogs, an increase in the percentage of lymphocytes, eosinophils, and neutrophils was detected. Sensitized dogs developed chronic interstitial inflammation with lymphocytes, macrophages, plasma cells, and eosinophils in lungs. Granulomas with lymphocytes, histiocytes, and giant cells were detected in both the interstitium and the bronchiolar wall in the lungs of sensitized dogs. PaO² was lowest in dogs showing the most severe interstitial inflammation in the lungs. The results indicate that dogs can be successfully used in immunologic and physiologic studies of PS-induced HP.     

The effect of CTLA-4Ig, a CD28/B7 antagonist, on the lung inflammation and T cell subset profile during murine hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease characterized by an influx of activated T cells to the lung, in which the CD28/B7 costimulatory signals are essential for the T cell activation and the outcome of the inflammatory response. In this study, we investigated the effect of the CD28/B7 antagonist, CTLA-4Ig, on the lung inflammation and the T cell subset profile in experimental Saccharopolyspora recivirgula (SR)-induced HP. C57BL/6 mice were treated with SR or saline during two and three weeks and in addition of CTLA-4Ig was administrated after either the second or third week and mice were sacrificed seven days later. The extent of the lung inflammation was quantified by histopathology and the lung T cell subsets (Treg, Th17, γδT and NKT) were analyzed by flow cytometry. Mice treated with CTLA-4Ig showed a significant decrease in the extent of lung damage (p < 0.05), and exhibited a decreased number of inflammatory cells in the bronchoalveolar lavage (BAL) with diminished CD4/CD8 T cell ratio. Also, a significant increase in the percentage of lung γδT (p < 0.01) and NKT (p < 0.05) cells was observed in two weeks SR-treated mice with the administration of CTLA-4Ig/SR. At 3 weeks, SR-treated mice showed an increased percentage of regulatory T cells but no significantly differences were found in the percentage of Th17 cells when compared with CTLA-4Ig/SR-treated mice. Our findings suggest that the treatment with CTLA-4Ig affects the HP progression and the lung T cell subset kinetics in mice.     

Treatment and monitoring of hypersensitivity pneumonitis

Introduction: Hypersensitivity pneumonitis (HP) is an immunologically induced lung disease that develops after inhalation of certain environmental antigens only in subjects with susceptibility to antigens. Therefore, both environmental and host immunological factors play important roles in the aetiology and pathogenesis of HP.

Areas covered: Determination of an inciting antigen is crucial for diagnosis, treatment and monitoring. For treatment, modification of the environment and of the host immune response are important. The former includes reduction of antigenic burden (i.e. disinfectant, cleaning), protective devices (i.e. filter, respiratory protection mask, ventilation) and avoidance of inciting antigens. The latter includes corticosteroids, lung transplantation and smoking cessation. For monitoring, measurement of serum Krebs von den Lungen (KL)-6 and surfactant protein (SP)-D concentrations can be used to screen for HP and to detect HP activity.

Expert commentary: Measurement of an inciting antigen may be useful to predict the progression and prognosis of the disease. Treatment and monitoring are challenging in chronic HP with fibrosis.     

Transporter associated with antigen processing (TAP) 1 gene polymorphisms in patients with hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a lung inflammatory disease caused by the inhalation of a variety of antigens. Previous studies support the role of the major histocompatibility complex (MHC) class II genes in the susceptibility to develop HP. However, the putative role of other MHC loci has not been elucidated. Transporters associated with antigen processing (TAP) genes are located within the MHC class II region and play an important role transporting peptides across the endoplasmic reticulum membrane for MHC class I molecules assembly. The distribution of single nucleotide polymorphisms (SNPs) in TAP1 genes was analyzed in 73 hypersensitivity pneumonitis (HP) patients and 58 normal subjects. We found a significant association of the allele Gly-637 (GGC) (p=0.00004, OR=27.30, CI=3.87-548.04) and the genotypes Asp-637/Gly-637 (p=0.01, OR=16.0, CI=2.19-631.21), Pro-661/Pro-661 (p=0.006, OR=11.30, CI=2.28-75.77) with HP. A significant decrease in the frequency of the allele Pro-661 (CCA) (p=0.008, OR=0.06, CI=0-0.45), the genotype Asp-637/Asp-637 (p=0.01, OR=0.17, 95% CI=0.05-0.58) and the haplotype [Val-333 (GTC), Val-458 (GTG), Gly-637 (GGC), Pro-661 (CCA)] was detected in HP patients compared with controls (p=0.002, OR=0.07, CI=0.0-0.57). These findings suggest that TAP1 gene polymorphisms are related to HP risk, and highlight the importance of the MHC in the development of this disease.     

Personal spore sampling and indirect immunofluorescent test for exploration of hypersensitivity pneumonitis due to mould spores

Sensitization to mould spores was investigated in six patients with hypersensitivity pneumonitis, eight patients with idiopathic lung fibrosis, and six healthy controls by immunodiffusion and immunofluorescent testing of personal spore samples. The new technique of personal spore sampling with the Burkard personal volumetric air sampler and indirect immunofluorescent test for detection of spore-specific IgG and IgM confirmed that five patients with hypersensitivity pneumonitis and four with lung fibrosis were actually exposed and sensitized to moulds. Personal spore sampling and subsequent immunofluorescent tests represent sensitive tools for detection of individual mould sensitization and air quality control.     

Distinct histopathology of acute onset or abrupt exacerbation of hypersensitivity pneumonitis

Hypersensitivity pneumonitis is an inflammatory lung disease that develops in response to exposure to antigen. Cases can be stratified by the duration of exposure and speed of symptom progression into acute, subacute, and chronic hypersensitivity pneumonitis. Although the pathologic features of subacute hypersensitivity pneumonitis are well established and those of chronic hypersensitivity pneumonitis have been reported, little is known about the histopathology of acute hypersensitivity pneumonitis. We evaluated the pathologic features of 5 patients with clinically confirmed hypersensitivity pneumonitis and rapid onset of symptoms and 3 patients with subacute or chronic hypersensitivity pneumonitis with symptom exacerbation. Histopathologic features assessed in each case included those characteristic of subacute hypersensitivity pneumonitis (bronchiolocentric chronic inflammation, histiocytic aggregates, and bronchiolitis obliterans), those associated with acute inflammation (fibrin deposition and neutrophilic infiltrate), and fibrosis. The classic features of hypersensitivity pneumonitis were identified in all 8 cases, with 1 also exhibiting fixed fibrosis confirming underlying chronic hypersensitivity pneumonitis. Fibrin deposition was present in 8 (100%) of 8 cases, and its extent was significant (28% surface area fibrin deposition/total disease area on average). Two had intra-alveolar fibrin so marked that it resembled acute fibrinous and organizing pneumonia. In addition, prominent interstitial neutrophilic infiltrate (≥5 cells/high-power field) was seen in all cases. These features have not been reported as characteristics of subacute or chronic hypersensitivity pneumonitis. Increased fibrin deposition and neutrophilic infiltrate may characterize acute hypersensitivity pneumonitis or abrupt exacerbation of hypersensitivity pneumonitis, and these along with characteristic features of subacute hypersensitivity pneumonitis (granulomatous inflammation and bronchiolocentricity) are sufficient to establish a morphologic diagnosis, particularly in conjunction with clinicoradiologic features.     

Immunoregulation in hypersensitivity pneumonitis. I. Differences in T-cell and macrophage suppressor activity in symptomatic and asymptomatic pigeon breeders

Cell-mediated immunity delineates symptomatic from asymptomatic pigeon breeders better than the presence of precipitating antibodies or HLA haptotypes. We therefore investigatedin vitro lymphocyte function in asymptomatic and symptomatic pigeon breeders. Peripheral blood, T, B, and adherent numbers were equal in both groups. Although the response to an optimum dose of concanavalin A was similar in both groups, the asymptomatic group revealed a reduced response to an optimum concentration of phytohemagglutinin and pokeweed mitogen. This decreased mitogen-induced proliferation was abrogated if cells from asymptomatic patients were precultured in medium alone for 48 hr before stimulation. A similar preincubation procedure, however, decreased the response to subsequent mitogenic stimulation in the symptomatic patients. As this suggested the possibility of heightened immunosuppression in the asymptomatic group, suppressor cells were studied. The numbers of FC-bearing T cells were similar in both groups and did not differ from control values. In addition, concanavalin A-induced suppressor-cell activity did not differ in either group. Nonetheless, fresh T cells, adherent cells, and peripheral blood mononuclear cells (PBMC) preincubated with pigeon serum decreased the response of preincubated autologous PBMC to mitogens and antigens in asymptomatics. A similar suppression did not occur in autoch-thonous cocultures of PBMC from symptomatic patients. These data suggest that there may be cellular suppressor influences in the asymptomatic group which are absent or nonfunctional in the symptomatic patients. Although this may be useful in distinguishing between the groups, the relationship of these suppressor cells, if any, to the pathogenesis of hypersensitivity pneumonitis awaits further study.     

IFN-gamma production by innate immune cells is sufficient for development of hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is an interstitial lung disease that develops following repeated exposure to inhaled particulate antigens. The disease is characterized by lymphocytic alveolitis, granuloma formation and fibrosis. IFN-gamma is required for the formation of granulomas in HP, and we therefore focused on identifying the cellular sources of IFN-gamma during the disease. Using the Saccharopolyspora rectivirgula (SR) animal model of HP, we demonstrated that the majority of IFN-gamma(+) cells in the lung following SR exposure are neutrophils. Ab-mediated depletion of neutrophils in mice prior to exposure to SR resulted in a decrease in the level of IFN-gamma mRNA and protein compared to isotype Ab-treated mice, suggesting that neutrophils are an important source of IFN-gamma during HP. To determine the contribution of T and non-T cell sources of IFN-gamma to granuloma formation, we performed adoptive transfer studies. RAG-1(-/-) mice reconstituted with spleen cells from IFN-gamma(-/-) mice developed granulomas similarly to RAG-1(-/-) mice reconstituted with normal spleen cells. Therefore innate immune cell IFN-gamma production in the absence of T cell IFN-gamma production is sufficient for granuloma formation. These results provide new insight into the pathogenesis of HP and demonstrate the important contribution of innate immune cells to the disease process.     

Anti-avian antibodies and rheumatoid factor in pigeon hypersensitivity pneumonitis

BACKGROUND: Although several immunological abnormalities may be present in pigeon hypersensitivity pneumonitis (HP), few specific hallmarks have been described. OBJECTIVE: To determine whether the presence of rheumatoid factor (RF) could be useful to discriminate pigeon HP from asymptomatic breeders (AB) and other interstitial lung diseases. METHODS: Fifty-three patients with pigeon HP, 47 AB, 31 idiopathic pulmonary fibrosis (IPF) patients and a rheumatoid arthritis (RA) group were studied. IgM RF was determined through enzyme-linked immunosorbent assay (ELISA) and western blot using human IgG and IgG Fc fragment as antigens. IgG and IgA anti-avian antibodies (AA) against pigeon serum antigen were also measured. The use of F(ab')2 fraction of peroxidase-labelled anti-human immunoglobulins prevented endogenous interferences. Possible cross-binding of RF with avian antigens and the reactivity against human IgG by AA were studied. RESULTS: RF tests were frequently positive in HP (52.8%) in comparison to AB (4.2%) and IPF (12.9%; P = 2.6 x 10-10 and 4.1 x 10-5). Therefore, the presence of RF in pigeon HP showed a sensitivity of 52% and was highly specific considering the results of AB and IPF (95 and 87%, respectively). The RA group revealed positive RF but negative AA tests. RF activity was confirmed through western blot using purified IgG Fc fragment. Overlapping levels of IgG and IgA AA were found in HP and AB. The frequency of AA was low in IPF. The cross-reaction of RF with avian antigens was excluded, and no reactivity against human IgG by AA was detected. Other endogenous interferences were ruled out. CONCLUSION: No single immunological test may definitively distinguish pigeon HP from AB and other interstitial lung disorders; however, positive RF, together with high AA levels, seems to be useful in differentiating the diagnosis.     

Histopathological spectrum of hypersensitivity pneumonitis with clinico‐radiologic correlation

There is no consensus on the classification of the diagnostic certainty of hypersensitivity pneumonitis (HP) based on the histopathological findings. This retrospective study aimed to describe the clinical and histopathological spectrum of HP. Herein, we also propose different grades of diagnostic certainty. Based on the histology, the cases were classified as: ‘definite HP’, ‘probable HP’, and ‘possible HP’. Of the 47 subjects screened, 30 cases of histologically diagnosed HP (mean age 48.4 years; 50% women) were included. The findings of cellular bronchiolitis, interstitial pneumonia, interstitial granuloma, isolated interstitial multinucleated giant cells (MNGCs), airspace granulomas, isolated airspace MNGCs, and organizing pneumonia were present in 96.7%, 80%, 46.7%, 50%, 10%, 63.3%, and 16.7% cases, respectively. Based on the various combinations of histopathological findings, the cases were classified as ‘definite’, ‘probable’, and ‘possible’ HP in 56.7%, 33.3%, and 10%, respectively. Chronic HP was diagnosed in 56.7% cases based on the presence of fibrosis on histopathology. The histopathological diagnosis of subacute or chronic HP did not corroborate with the disease duration, and 17.6% of the subjects with duration of symptoms of <6 months had evidence of fibrotic disease on histopathology.     

Pulmonary alveolar macrophages in patients with sarcoidosis and hypersensitivity pneumonitis: Characterization by monoclonal antibodies

Using a panel of monoclonal antibodies (MoAbs), the frequency of cells bearing Class I and Class II major histocompatibility complex (MHC) determinants, transferrin receptor (TR) sites, and interleukin-2 receptors (IL-2R) has been evaluated on pulmonary alveolar macrophages (PAM) recovered from the bronchoalveolar lavage (BAL) fluid of 21 patients with pulmonary sarcoidosis (including 11 cases with active sarcoidosis and 10 cases with inactive disease), 8 patients with hypersensitivity pneumonitis (HP), and 6 normal non-smoking volunteers. When the frequency of Class II DR-positive cells was considered, 64.3% of control PAM expressed HLA-DR products. No statistically significant differences were observed between controls and sarcoid patients, while HP patients showed an enhanced proportion of DR⁺ PAM with respect to normal PAM (P<0.05). On the contrary, the frequency of PAM expressing HLA-DQ molecules was higher in both active sarcoidosis and HP patients with respect to patients with inactive sarcoidosis and normal subjects (P<0.001). A statistically significant increase in Class I antigen-positive PAM has been demonstrated in HP patients as compared to controls (P<0.05). Active sarcoid patients showed a higher number of PAM-bearing TR sites than controls and other groups of patients considered (P<0.001). An increase in the percentage of IL-2R-positive PAM has been demonstrated in active sarcoidosis (P<0.001). Our data suggest that (1) PAM of patients with the above-considered interstitial lung diseases are in a state of activation and exhibit structures which play a crucial role in antigenic recognition by T lymphocytes, such as HLA-DQ molecules; (2) the presence of TR in PAM of patients with active sarcoidosis could be related to a more advanced differentiation stage of these cells and/or to particular functional properties; and (3) a direct role of the IL-2/IL-2R system in the interaction between T cells and monocytes in sarcoid lung is crucial. Besides representing an additional parameter which differentiates BAL features of sarcoidosis from those of HP patients, these results could represent a useful tool in the evaluation of the macrophagic component of alveolitis by the BAL.     

Bronchiolitis interstitial pneumonitis: a pathologic study of 31 lung biopsies with features intermediate between bronchiolitis obliterans organizing pneumonia and usual interstitial pneumonitis, with clinical correlation

Bronchiolitis combined with interstitial pneumonitis generally has been equated with bronchiolitis obliterans organizing pneumonia (BOOP). We describe our experience with lung biopsies that had both bronchiolar and interstitial diseases. We studied 31 patients who had respiratory difficulty leading to open lung biopsy, which showed a combination of both prominent bronchiolitis and prominent interstitial pneumonitis. We compared these cases clinically and pathologically with 6 other pulmonary diseases, namely, bronchiolitis obliterans, BOOP, nonspecific interstitial pneumonitis, usual interstitial pneumonitis, airway-centered interstitial fibrosis, and idiopathic bronchiolocentric interstitial pneumonia, and with 10 cases of cystic fibrosis, an unrelated disease with both bronchiolar and interstitial pathology. The commonality of our cases was a combination of bronchiolitis and interstitial inflammation and fibrosis but little or no intra-alveolar organizing pneumonia. Bronchiolitis obliterans with organizing pneumonia involved less area than the interstitial pneumonitis in each case. All 19 patients for whom we had follow-up received corticosteroids for their pulmonary diseases. Seven patients had improvement in symptoms and pulmonary function test results and radiographic findings, 5 patients experienced subjective improvement with unchanged results of pulmonary function tests or chest x-ray, 1 patient's condition was unchanged, 6 patients' disease worsened, and 4 of these 6 died. The natural history of these cases, which we have designated bronchiolitis interstitial pneumonitis, seems more sanguine than usual interstitial pneumonitis and worse than BOOP at least in the short term. On the one hand, response to corticosteroids was not as frequent as generally accepted for BOOP. On the other hand, disease did not progress in most patients on corticosteroids.     

A case of hypersensitivity pneumonitis caused by Penicillium species in a home environment

We report a case of hypersensitivity pneumonitis in a 30-yr-old female housewife caused by Penicillium species found in her home environment. The patient was diagnosed according to history, chest radiograph, spirometry, high-resolution chest CT, and transbronchial lung biopsy. To identify the causative agent, cultured aeromolds were collected by the open-plate method. From the main fungi cultured, fungal antigens were prepared, and immunoblot analysis with the patient's serum and each fungal antigen was performed. A fungal colonies were isolated from the patient's home. Immunoblotting analysis with the patient's sera demonstrated a IgG-binding fractions to Penicillium species extract, while binding was not noted with control subject. This study indicates that the patient had hypersensitivity pneumonitis on exposure to Penicillium species in her home environment.     

Chronic hypersensitivity lung disease with recurrent episodes of hypersensitivity pneumonitis due to a contaminated central humidifier

A child with a 4-year history of acute and chronic respiratory symptoms of unknown aetiology was investigated for hypersensitivity pneumonitis. Lung disease due to inhalation of material from a contaminated central humidifier was suggested by the clinical history, the presence of precipitating antibodies in the serum against the humidifier water, a pulmonary response to challenge with the humidifier water, and marked improvement after removal of the humidifier. No fungi were cultured from the humidifier nor were antibodies against a number of fungal antigens identified by radioimmunoassay inhibition techniques. Antigenic material was found in the humidifier water and the household water prior to its reaching the humidifier. This antigenic material was not found in laboratory tap water supplied from the same general source (Lake Michigan) but from a different pumping station. Three of the child's siblings gave histories suggestive of a single concurrent episode of acute hypersensitivity pneumonitis and one sibling had a history suggestive of chronic hypersensitivity lung disease. No association could be found between HLA-haplotypy and disease in the patient and the siblings.     

Detection of IgM antibodies against cytomegalovirus: comparison of two radioimmunoassays, enzyme-linked immunosorbent assay and immunofluorescent antibody test

The sensitivity and specificity of direct antibody radioimmunoassay (RIA), M-antibody capture RIA (MACRIA), enzyme-linked immunosorbent assay (ELISA), and the immunofluorescent antibody (IFA) test for the detection of CMV-specific IgM was compared using 40 sera selected from different groups of patients. RIA, MACRIA, and ELISA gave concordant results with thirty-two sera but discordant results with eight sera, of which three were cord sera from congenitally infected babies, three were from immunocompromised patients with recurrent CMV infections, and two were from patients with lymphadenopathy and Paul-Bunnell-positive mononucleosis, respectively. RIA, MACRIA, and ELISA were of similar sensitivity with sera from adult patients, but ELISA was apparently less sensitive than RIA and MACRIA for the detection of CMV IgM in cord serum. By comparison IFA was significantly less sensitive than the other three tests. Rheumatoid factor is reactive in RIA, ELISA, and IFA but can efficiently be removed by absorption with latex-IgG beads or cross-linked human IgG.