Prolonged exposure to M. faeni in strain II guinea-pigs: pulmonary interstitial inflammation.

Models of hypersensitivity pneumonitis (HP) should exhibit progression of pulmonary histological abnormalities during continuing challenges. Strain II guinea-pigs were sensitized with Micropolyspora faeni and received 2, 4, or 8 weekly intratracheal (i.t.) particulate M. faeni challenges. Control animals received normal saline (NS). Four days after the last exposure, randomly selected microscopic fields of lung (200/animal) were judged to be normal or abnormal. If abnormal, the location and nature of the abnormalities were determined. Compared with NS treated guinea-pigs, those exposed to 2, 4 and 8 weekly M. faeni challenges exhibited more extensive (P less than 0.001) pulmonary histological abnormalities which involved both the intraalveolar and interstitial compartments. More extensive abnormalities in the 8 week group compared with the 4 week group were caused by increased extent of interstitial mononuclear cell infiltration. The extent of pulmonary interstitial histological abnormalities transiently (four challenges) decreases, but then increases, so that progressive pulmonary inflammation occurs during continuing challenges.     

Alveolar macrophage catabolism of Micropolyspora faeni

Pulmonary histologic abnormalities resolve despite continuing intratracheal injections of Micropolyspora faeni in a rabbit model of hypersensitivity pneumonitis. We examined in vitro alveolar macrophage (AM) metabolism to determine if increased efficiency of M. faeni degradation by AMs was associated with resolution of pulmonary abnormalities. Rabbits were exposed to M. faeni with three sensitizing and two, four, or eight weekly intratracheal challenge injections. Bronchoalveolar cells (BAC) were obtained by lavage 4 to 6 days after the last intratracheal injection. We determined the fate of 125I-labeled M. faeni added to 48-hour cultures of BAC derived from naive and M. faeni-exposed animals. Label was transported from the pellet to the supernatant fraction of BAC cultures, and the proportion of supernatant label that was precipitated by trichloroacetic acid decreased. These phenomena were dependent on time, viable cells, and temperature. They were not altered by puromycin and were caused by AM. BAC from M. faeni-treated rabbits were slightly more effective in transport of label from pellet to supernatant than BAC from naive rabbits during the first 4 hours of culture but not thereafter. There was no difference between BAC from rabbits challenged two, four and eight times. We conclude that resolution of pulmonary histologic abnormalities in this model of hypersensitivity pneumonitis is not associated with evidence of enhanced AM particulate M. faeni catabolism.     

Mast cell hyperplasia in experimental hypersensitivity pneumonitis.

We investigated the presence of mast cells in a model of experimental hypersensitivity pneumonitis (EPH). Guinea pigs exposed to 8 weekly intratracheal challenges with Micropolyspora faeni exhibited significant increases in the number of mast cells within the lung as compared to controls and animals challenged only 2 or 4 times. The number of cells in M. faeni-challenged animals were increased around bronchi, bronchioles, blood vessels and in alveolar septa. There appeared to be contraction of peribronchial, peribronchiolar and vascular smooth muscle. Ultrastructural examination of lung tissue revealed the presence of degranulating mast cells. Bronchoalveolar lavage histamine levels were increased after 8 but not after 2 or 4 weekly challenges. Serum anti-M. faeni antibody was present in all M. faeni-exposed animals but not in control animals. We conclude that mast cells and histamine levels in bronchoalveolar lavage fluid are increased in a model of EHP caused by repetitive, intratracheal injection of M. faeni particulate antigen.     

The effect of cortisone acetate on pulmonary lesions in a rabbit model of hypersensitivity pneumonitis (cortisone and hypersensitivity pneumonitis).

Rabbits were sensitized with Micropolyspora faeni by intratracheal inoculations and later challenged with the same antigen either with or without parenteral administration of cortisone acetate prior to challenge. Animals developed anti-M. faeni serum precipitins, M. faeni-induced alveolar macrophage migration inhibition, and positive 48-hr skin reactivity to M. faeni. Sensitized animals also demonstrated an augmented pulmonary histopathological response following respiratory challenge with M. faeni when compared to non-sensitized controls. Cortisone acetate abrogated this augmented pulmonary histopathological response following challenge with M. faeni. Cortisone acetate also abolished the positive alveolar macrophage migration inhibition found in sensitized animals.     

Comparison of allergic aspermatogenesis with that induced by vasectomy. I. In vivo studies in the guinea-pig.

Groups of male and female guinea-pigs were immunized with homologous epdidymal sperm in Freund's complete adjuvant (FCA) and skin tested at weekly intervals with a heat-treated extract of the sperm or with PPD. In females, the skin response to both antigens was similar to that to any standard protein antigen. In males, the response to sperm extract varied with the induced auto-immune orchitis, reaching a maximum immediately after testis lesion was most severe and as recovery was beginning (shown histologically): the response to PPD was decreased at 1 week after immunization, but subsequently was similar to that in females. Skin tests on guinea-pigs 8 months after bilateral vasectomy (when both ends of vasa were ligated), showed no evidence of delayed hypersensitivity to sperm, although there was marked histological evidence of reduced spermatognesis, due to back pressure atrophy.     

Macrophage activation by Micropolyspora faeni does not suppress anamnestic pulmonary immunologic reactivity

Determinants of lung immunologic response to antigens are not known, but could include alveolar macrophage (AM) activation. We tested the ability of AM activation to modify the anamnestic response by administering bovine serum albumin (BSA) intratracheally, activating AM (by intratracheal Micropolyspora faeni), and then exposing rabbits again to intratracheal BSA. We compared the results from 4 groups of animals: intratracheal administration of either 50 mg M. faeni or normal saline and later administration of either intratracheal or intramuscular BSA. M. faeni administered intratracheally increased the number of AM. These AM were activated (increased phagocytosis of IgG-coated particles). We found no difference in the amount of antibody in either lavage fluid or serum or in antigen-induced pulmonary parenchymal and hilar node lymphocyte proliferation among these 4 groups.     

Hypersensitivity pneumonitis: whole Micropolyspora faeni or antigens thereof stimulate the release of proinflammatory cytokines from macrophages

Hypersensitivity pneumonitis (HP) is an allergic granulomatous interstitial lung disease resulting from a reaction of selected individuals to repeated inhalations of certain antigens. HP is characterized by chronic inflammation, and the development of the disease seems to be immunologically mediated. In farmer's lung, the source of provoking antigen has been found to be actinomycetes such as Micropolyspora faeni. In this study, we show that M. faeni, or antigens thereof, stimulate strong release of proinflammatory cytokines from blood monocytes and alveolar macrophages obtained from nonfarmer volunteers and naive mouse peritoneal macrophages. Interleukin-1 (IL-1) was produced by human alveolar macrophages and murine peritoneal macrophages in response to whole M. faeni and antigens thereof. IL-1 activity was detected in the supernatants at 12 h of incubation and was maximal by 24 to 36 h (200 to 400 U/ml of IL-1). A rabbit antiserum to IL-1 alpha and IL-1 beta neutralized the thymocyte-stimulating activity of the supernatants. Moreover, M. faeni (1 to 100 micrograms of antigen) elicited a strong secretion of tumor necrosis factor-alpha (TNF-alpha) from human alveolar macrophages and monocytes as well as mouse peritoneal macrophages, where 1 micrograms of M. faeni elicited the secretion of approximately 100 U of TNF-alpha from 2 x 10(5) macrophages, and 100 micrograms stimulated the release of approximately 1,000 U of bioactive TNF-alpha. One particle of whole M. faeni per cell was sufficient to induce copious release of TNF-alpha from macrophages or monocytes (100 U of bioactive TNF-alpha; 1,000 pg/ml of antigenic TNF-alpha as seen by radioimmunoassay). Both IL-1 and TNF-alpha productions stimulated by M. faeni were not abrogated by inclusion of polymyxin B. We propose that the direct stimulation of cytokines by M. faeni or antigens thereof may play an important role in HP.     

The value of classifying interstitial pneumonitis in childhood according to defined histological patterns

Aims : Interstitial pneumonitis in children is very rare and most cases have been classified according to their counterparts in adults, although the term 'chronic pneumonitis of infancy' has recently been proposed for a particular pattern of interstitial lung disease in infants. We reviewed our paediatric cases of interstitial pneumonitis, first, to look at the spectrum of histological patterns found in this age group and, second, to determine whether the classification of such cases in childhood is both appropriate and worthwhile.  

Methods and results

Twenty-five of 38 open lung biopsies showed an overlapping spectrum of interstitial pneumonitis, including three cases that fulfilled the histological criteria for chronic pneumonitis of infancy. There were 11 cases of reactive pulmonary lymphoid hyperplasia (either lymphoid interstitial pneumonitis or follicular bronchiolitis), five of which were associated with abnormalities of the immune system. Four cases were classified as desquamative interstitial pneumonitis and the remaining seven cases were classified as non-specific interstitial pneumonitis. There were no cases with the histological features of usual interstitial pneumonitis. Most patients responded to steroids but tended to have a residual deficit in lung function. Mortality appeared to be associated with presentation at a young age.  

Conclusion

Classification of interstitial pneumonitis according to their adult counterparts is appropriate for this younger age group and can provide valuable information for the clinician. The term 'chronic pneumonitis of infancy' refers to a specific histological pattern, but whether it represents a separate disease or a reflection of pulmonary immaturity remains to be proven.     

Antigens of Micropolyspora faeni strains.

Exocellular antigens from Micropolyspora faeni and Micropolyspora rectivirgula were prepared by growing them in a synthetic broth culture at 50 degrees C for 1 week under continuous shaking. The broth was separated from the organisms by filtration and the filtrate was dialyzed and freeze-dried. The cross-reactivity of these antigens were studied by antigen-antibody crossed-immunoelectrophoresis and the presence of similar antigens in different strains by tandem crossed-immunoelectrophoresis using rabbit anti-M. faeni and anti-M. rectivirgula sera. The results indicate that a number of antigenic components are common to the different strains of M. faeni and M. rectivirgula, while each strain demonstrates several specific antigenic determinants. Of the 26 clearly demonstrable precipitin arcs formed by crossed-immunoelectrophoresis, only 1 was detected in all the strains while 2 more were detected frequently. This study indicates that M. rectivirgula and M. faeni strains are not distinguishable from each other by morphological, biochemical or immunological criteria.     

Embolic pneumopathy induced by oleic acid. A systematic morphologic study.

This paper presents a systematic study of acute and chronic pulmonary lesions resulting from a single intravenous injection of oleic acid and a new fibrosis lung model is proposed: pulmonary interstitial fibrosis is obtained by means of a number of oleic acid intravenous injections. Nineteen adult dogs received 0.045 g/kg or 0.09 g/kg of oleic acid. A systematic morphologic study was carried out after 1, 2, 3, 4, 6, 12, 24, and 48 hours and 1, 2 and 4 weeks. Eleven other adult dogs received weekly one injection of 0.09 g/kg of pure oleic acid over a period of 1 to 3 months. Examination of the lung was carried out by means of light and electron microscopy and morphometry. An early stage characterized by the formation of thrombosis and cellular necrosis was followed by a repair stage with the proliferation of Type 2 cells and fibrotic foci in the subpleural areas. Lipid staining with Sudan IV allowed the onset and disappearance of lipid-laden macrophages to be ascertained. The late stage showed pulmonary fibrosis. The extent of the lesions is related to the number of oleic acid injections. Since interstitial pulmonary fibrosis invariably appeared, and only 2 dogs out of 11 died, the model is satisfactory for pathologist and physiologist.     

T-lymphocyte effects on murine cytomegalovirus pulmonary infection.

Cytomegalovirus (CMV) infections were induced in male BALB/c mice treated with rat monoclonal antibodies (MAb) to deplete selectively CD8 and CD4 cell populations in vivo. The animals were then inoculated intraperitoneally with murine CMV and the infection was monitored virologically and histologically. High concentrations of virus were found in the lungs of mice depleted of CD4 or both CD4 and CD8 cells. These animals developed pulmonary infections that persisted for at least 49 days after inoculation. In contrast, immunologically intact mice and those administered anti-CD8 MAb experienced only a transient infection of the lungs. Focal interstitial infiltrates of mononuclear cells were demonstrated in pulmonary tissues of CD4 MAb-treated animals, but not in normal mice and those receiving the CD8 MAb. Adoptive transfer of CD4 cells to animals (rendered immune-incompetent by thymectomy and irradiation) protected against pulmonary infection and the development of interstitial pneumonia. Mice treated with CD4 MAb failed to produce specific CMV antibody, whereas the depletion of CD8 cells had no effect on antibody elaboration. Administration of anti-CD4 and CD8 MAb did not affect virus replication in the salivary glands, the preferential site for CMV infection in the mouse. Induction of pulmonary infection and interstitial pneumonia by CMV in BALB/c mice is mediated by CD4 T cells.     

Effects of alpha-tocopherol on cadmium-induced toxicity in rat testis and spermatogenesis

Cadmium is known to exert toxic effects on multiple organs, including the testes. To determine if alpha-tocopherol, an antioxidant, could protect testicular tissues and spermatogenesis from the toxic effects of cadmium, six-week old male Sprague-Dawley rats were randomized to receive cadmium at doses of 0 (control), 1, 2, 4 or 8 mg/kg by the intraperitoneal route (Group A) or alpha-tocopherol for 5 days before being challenged with cadmium (Group B) in an identical dose-dependent manner. When both groups received cadmium at 1 mg/kg, there were no changes in testicular histology relative to controls. When Group A received cadmium at 2 mg/kg, undifferentiated spermatids and dead Sertoli cells increased in the seminiferous tubules while interstitial cells decreased and inflammatory cells increased in the interstitial tissues. On flow cytometric analysis, the numbers of elongated spermatids (M1) and round spermatids (M2) decreased while 2c stage cells (M3, diploid) increased. In contrast, when Group B received cadmium at 2 mg/kg, the histological insults were reduced and the distribution of the germ cell population remained comparable to controls. However, alpha-tocopherol had no protective effects with higher cadmium doses of 4 and 8 mg/kg. These findings indicate that alpha-tocopherol treatment can protect testicular tissue and preserve spermatogenesis from the detrimental effects of cadmium but its effectiveness is dependent on the dose of cadmium exposed.     

Chromosome 16 abnormalities associated with myeloid malignancies

Twenty-six patients who had cytogenetic analyses performed for myeloid malignancies at St. Vincent's Hospital over a 6-year period were found to have an inversion abnormality of chromosome 16 (25 patients) or t(16;16) (1 patient). Only 16 patients had all the features of M4Eo, while the other 10 patients had diagnoses of M2, M4, M5, RAEB, and RAEB-T; six of these had abnormal eosinophils. Thus, abnormal eosinophils were present in 22 of 26 patients (85%). Thirteen patients had additional cytogenetic abnormalities at diagnosis, the commonest being +8 in 5, del(7q) in 4, and +21 in 3. Twenty-three patients received chemotherapy and 20 (87%) achieved complete remission. The median survival of the treated group was 188 weeks with a 61% 2-year and 45% 5-year survival. No significant difference in survival was observed between those patients with a diagnosis of M4Eo and those with other diagnoses suggesting that it is the abnormality of chromosome 16 which confers an improved prognosis. Additional cytogenetic abnormalities present at diagnosis did not affect prognosis. CNS relapse was observed in only two patients (8%), thus indicating no increased incidence of this complication. This study supports the premise that a chromosome abnormality involving 16p13 and 16q22 defines a good prognosis subset of myeloid leukemia despite morphological variations.     

Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis.

Silica-induced pulmonary fibrosis usually follows exposure to increased levels of this particulate and its retention in interstitial macrophages of the lung. It is suggested that accelerated clearance of particles from the pulmonary interstitium may ameliorate subsequent fibrosis. To test this hypothesis, one group of mice received 2-mg intratracheal (IT) silica; some particles were phagocytized and cleared during the subsequent inflammatory response, other particles were translocated across the epithelium to reach interstitial macrophages by 2 weeks. These mice later showed increased fibroblast growth, a doubling of lung collagen levels and large silicotic nodules by 16 weeks when much of the silica was still present in the lung. A second group of mice received IT silica, then 2 and 3 weeks later received IT injections of N-formyl-L-methionyl-leucyl-phenylalanine (FMLP), a leukocyte chemoattractant. Subsequently, a significant increase in inflammatory cells was seen and silica was observed mostly in phagocytes within the alveolar spaces. Few interstitial particles were found at 4 weeks, and extensive fibrosis did not develop by 16 weeks; only a few small nodules were seen and little silica was present in the lung. The results indicate that clearance of interstitial particles by a controlled inflammatory response is possible, and that removal of silica from the interstitium decreases the fibrotic response.     

Failure of macrophage activation to induce pulmonary fibrosis in asbestos-exposed guinea-pigs

Inhalation of asbestos by specific-pathogen-free (SPF) guinea-pigs the macrophages of which were systemically activated with Freund's complete adjuvant and/or M. tuberculosis (Strain H37Ra) induced a diffuse pulmonary mononuclear-cell infiltration of the interstitium and air space. The severity of the reactions to the various insults was dependent on the treatment given. Although there was no histological evidence of increased collagen, there was a diffuse increase in reticulin in animals which were exposed to asbestos and the macrophages of which were activated. However, even in the case where severe pulmonary inflammatory changes occurred complete resolution of the response took place within 1 year. Therefore in this model activation of macrophages does not have a detrimental effect after inhalation of asbestos.     

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.     

Failure of macrophage activation to induce pulmonary fibrosis in asbestos-exposed guinea-pigs.

Inhalation of asbestos by specific-pathogen-free (SPF) guinea-pigs the macrophages of which were systemically activated with Freund''s complete adjuvant and/or M. tuberculosis (Strain H37Ra) induced a diffuse pulmonary mononuclear-cell infiltration of the interstitium and air space. The severity of the reactions to the various insults was dependent on the treatment given. Although there was no histological evidence of increased collagen, there was a diffuse increase in reticulin in animals which were exposed to asbestos and the macrophages of which were activated. However, even in the case where severe pulmonary inflammatory changes occurred complete resolution of the response took place within 1 year. Therefore in this model activation of macrophages does not have a detrimental effect after inhalation of asbestos.     

盐皮质激素受体阻断剂对SiO_2诱导的肺巨噬细胞表型偏移的调节

目的:研究盐皮质受体阻断剂螺内酯(spirolactone,SPI)对二氧化硅(SiO_2)诱导的肺巨噬细胞表型偏移的影响。方法:将60只C57BL/6小鼠随机分为生理盐水(NS)组、SiO_2组、SPI治疗组(SiO_2+SPI组)及溶剂对照组(SiO_2+NS组),经口咽吸入SiO_2悬浊液(40 mg/kg)建立矽肺模型,对照组给予等量生理盐水,SiO_2+SPI组在造模后每天经口灌胃给予SPI(10 mg·kg~(-1)·d~(-1)),SiO_2+NS组以相同方式给予等量生理盐水。造模后3 d、14d、28 d取材,进行肺泡灌洗,取灌洗液细胞进行定量、分类计数及流式细胞术分析,右下肺叶用酶解法制成单细胞悬液行流式细胞术分析。结果:与SiO_2组相比,SPI治疗可以明显降低SiO_2干预后3 d、14 d支气管肺泡灌洗液总细胞数及巨噬细胞、中性粒细胞及淋巴细胞数。SiO_2干预后14 d、28 d,肺泡巨噬细胞和间质巨噬细胞由M1表型向M2表型偏移,SPI治疗可以有效逆转SiO_2诱导的巨噬细胞表型偏移。结论:SPI可以减轻SiO_2导致的肺泡炎症细胞浸润,可能是通过逆转SiO_2诱导的巨噬细胞表型偏移实现的。     

Experimental hypersensitivity pneumonitis: cellular requirements

We previously demonstrated that Thy1.2⁺, CD4⁺, Ia⁻ T cells are responsible for transfer of murine adoptive experimental hypersensitivity pneumonitis (adoptive EHP). To characterize the culture conditions necessary for development of these cells, we depleted cell cultures of Thy1.2⁺, CD4⁺, CD8⁺, or Ia⁺ cells using MoAbs and complement or magnetic beads, prior to culture of sensitized C3H/HeJ murine spleen cells (SC) with Micropolyspora faeni. After culture, cells were transferred to recipients which were later challenged intratracheally with M. faeni. The extent of pulmonary inflammatory changes in these animals was determined 4 days after intratracheal (i.t.) challenge with M. faeni. Cultured M. faeni-sensitized SC which had been treated before culture with media, complement only, anti-CD8 plus complement or magnetic beads alone could transfer EHP to naive animals. SC treated with anti-Thy1.2 or anti-CD4 plus complement could not transfer EHP. Treatment of SC with anti-Ia^k plus magnetic beads diminished the ability of cultured cells to transfer EHP. We conclude that the ability to produce cells able to adoptively transfer EHP is dependent on the presence of Thy1.2⁺, CD4⁺ and Ia⁺ cells, but not CD8⁺ cells, at the onset of culture.     

Histopathological and immunohistological changes in the rabbit lung after experimental exposure to a purified enzyme of Micropolyspora faeni.

Rabbits were exposed intratracheally to enzyme 1, a highly immunogenic esterase isolated from Micropolyspora faeni. A single exposure to active enzyme 1 induced no histologically or immunohistochemically detectable changes in the lungs of experimental animals, while signs of focal interstitial and perivascular inflammatory reactions were evident following a course of three exposures to the enzyme. Interstitial pneumonia with characteristic generalized vasculitis and perivasculitis was produced following seven or nine inoculations. An enzymatically inactive preparation of enzyme 1, even by repeated administration, proved ineffective in eliciting pneumonia. Intracellular antigen within macrophages/histiocytes was demonstrated in the lungs of all experimental animals, including those which had been exposed to inhibited enzyme. Repeated exposure to the enzymatically active preparation resulted in the deposition of immunoglobulin and complement in association with vascular endothelia and in the walls of small- and medium-sized blood vessels; both immunoglobulin and complement could also be demonstrated within macrophages/histiocytes. On the basis of these findings it is concluded that (1) Farmer's lung-like interstitial pneumonia may be produced in rabbits by exposure to a purified, enzymatically active derivative of M. faeni, (2) an important pathogenic principle of the disease may consist in the rapid vascular deposition of immune complexes (type III reaction), and (3) damage by direct enzyme action may prove to contribute significantly in eliciting tissue damage by (an) ancillary mechanism(s) not yet understood.