Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens.

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.     

Immunocytochemical observation of paraquat-induced alveolitis with special reference to class II MHC antigens

The expression of class II major histocompatibility complex (MHC) antigens on alveolar epithelial cells and macrophages was investigated immunocytochemically in paraquat-induced alveolitis in the rat lung. Until 2 days after paraquat injection, class II MHC antigens were expressed on the type II alveolar epithelium without any inflammatory cellular infiltration. From the 4th to the 7th day after paraquat injection, class II MHC antigen-positive macrophages increased in the alveolar spaces, whereas the expression on the type II alveolar epithelium became obscure. Over 10 days after the injection, interstitial fibrosis progressed and the intra-alveolar inflammatory infiltrates decreased. Epithelial cells lining the thickened fibrous septa no longer expressed class II MHC antigens. These results suggest that chemical stimuli can induce class II MHC antigen expression on the type II alveolar epithelium in the early stage of cellular injury, followed by inflammatory infiltration and interstitial fibrosis.     

Expression of the immunomodulator IL-10 in type I pneumocytes of the rat: alterations of IL-10 expression in radiation-induced lung damage.

Fibrosing alveolitis is a disease with inflammatory, proliferative, and fibrotic components. In different models, it has been shown that the cytokine interleukin-10 (IL-10) plays a conflicting role in inflammation-associated fibrotic processes, inasmuch as it is an anti-inflammatory cytokine but also a TH2 cytokine with inherent pro-fibrotic effects. IL-10 is produced primarily by inflammatory cells. In this report, we show in a rat model of radiation-induced fibrosing alveolitis that IL-10 is also produced by type I alveolar epithelial cells in both normal and fibrotic lungs. The total amount of IL-10 in the lung is increased after irradiation, but type I pneumoyctes contain less IL-10. The R3/1 permanent type I pneumocyte cell line also contains IL-10, which is reduced after irradiation. Whereas in the normal lung, the entire alveolar surface is covered by IL-10-producing pneumocytes, this continuity is interrupted in fibrotic lungs, because type I pneumocytes lack full differentiation and thus full spreading over the alveolar surface. The exposure of the IL-10-negative epithelial basal membrane may allow for an easier attachment of inflammatory cells such as alveolar macrophages. These cells have the potential to act in a pro-inflammatory way by tumor necrosis factor alpha and also in a pro-fibrotic way by activating TH2 cytokines.     

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.     

Injury,inflammation, and remodeling in fetal sheep lung after intra-amniotic endotoxin

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intra-amniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung.     

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant

Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freund's complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node.The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.     

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes

Background

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

Methods

We have compared by flow cytometry the surface expression of the major markers involved in the immunological synapse on the A549 cell line, the most popular model of type II alveolar epithelial cells, and freshly isolated cells. HLA-DR, CD80, CD86, ICOS-L, CD54, CD58 surface expression were studied in resting conditions as well as after IFN-γ/TNF-α treatment, two inflammatory cytokines, known to modulate some of these markers.

Results

The major difference found between the two cells types was the very low surface expression of HLA-DR on the A549 cell line compared to its constitutive expression on freshly isolated AECII. The surface expression of co-stimulatory molecules from the B7 family was very low for the CD86 (B7-2) and ICOS-L (B7-H2) and absent for CD80 (B7-1) on both freshly isolated cells and A549 cell line. Neither IFN-γ nor TNF-α could increase the expression of these classical co-stimulatory molecules. However CD54 (ICAM-1) and CD58 (LFA-3) adhesion molecules, known to be implicated in B7 independent co-stimulatory signals, were well expressed on the two cell types.

Conclusions

Constitutive expression of MHC class I and II molecules as well as alternative co-stimulatory molecules by freshly isolated AECII render these cells a good model to study antigen presentation.     

In vivo autoradiographic demonstration of beta-adrenergic binding sites in adult rat type II alveolar epithelial cells

Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (QNB) or the beta-adrenergic probe 3H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 micron sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In contrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of beta-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific beta-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of beta-adrenergic agonists on alveolar type II cells.     

Temporal expression of alpha-smooth muscle actin and drebrin in septal interstitial cells during alveolar maturation.

In rat lung, the definitive alveoli are established during development by the outgrowth of secondary septa from the primary septa present in newborn; however, the mechanism of alveolar formation has not yet been fully clarified. In this study, we characterize the septal interstitial cells in developing alveoli. During the perinatal period, alpha-SMA-containing slender cells were found in the primitive alveolar septa. Alpha-SMA-containing cells were detected at the tips of the septa until postnatal day 21, when the alveolar formation was almost completed, but disappeared in adult. Immunoelectron microscopy demonstrated that alpha-SMA is localized mainly in the cellular protrusions, which are connected with the elastic fibers around the interstitial cells. Developmentally regulated brain protein (drebrin) is also located in the cell extensions containing alpha-SMA in immature alveolar interstitial cells. In adult lung, alpha-SMA-positive cells are located only at the alveolar ducts but are not found in the secondary septa. Desmin is expressed only in alpha-SMA-containing cells at the alveolar ducts but not in those at the tip of alveolar septa. These results suggest that a part of the septal interstitial cells are temporarily alpha-SMA- and drebrin-positive during maturation. Alpha-SMA- and drebrin-containing septal interstitial cells (termed septal myofibroblast-like cells) may play an important role in alveolar formation.     

Cytotoxic effects of 3-methylindole on alveolar epithelial cells and macrophages: with special reference to microtubular and filamentous assemblies in alveolar type I cells of bovine lung

The alveolar type I cell is a major permeability barrier between the pulmonary interstitium and alveolar spaces and its thin cytoplasmic processes are greatly susceptible to injury. These cells are often observed to undergo progressive vesiculation, vacuolization and desquamation during 3-methylindole (3MI)-induced acute pulmonary edema after oral administration in goats and cattle. The present study describes proliferation of SER and the presence of polymerized tubulin in the form of microtubules arranged in large bundles shown at ultrastructural level as well as with immunofluorescence staining for tubulin in alveolar type I cells 72 hours after 3MI treatment. Such changes were not seen in pulmonary endothelial cells, alveolar type II cells, alveolar macrophages and neutrophils. The possible role of microtubules in alveolar type I cells as a mechanistic support to resist disruption against the forces of interstitial and alveolar edema is compared with alveolar type II cells, alveolar macrophages and neutrophils. The latter cells undergo dynamic movements in response to inflammatory stimuli and therefore did not show microtubules in their cytoplasm.     

Enhanced expression of Duffy antigen in the lungs during suppurative pneumonia.

Duffy antigen is a chemokine binding protein expressed on the surface of erythrocytes and postcapillary venular endothelial cells. It binds selective CXC and CC chemokines with high affinity. Although Duffy antigen is present in the normal pulmonary vascular bed, it is not known whether its expression is altered by innate inflammatory responses in the lungs. We studied Duffy antigen expression by immunohistochemistry in autopsy lung specimens from 16 cases of suppurative pneumonia, 11 cases of acute lung injury, and seven normal lungs. In lungs with suppurative pneumonia, Duffy antigen was expressed in higher numbers of pre- and postcapillary parenchymal vessels compared to normal specimens or specimens with acute lung injury (p<0.03 and p<0.02, respectively). Lungs with suppurative pneumonia also showed Duffy antigen expression on the alveolar septa, whereas this was a rare finding in normal specimens or in acute lung injury (p<0.02). Furthermore, Duffy antigen labeling of the alveolar septa localized to regions with airspace accumulation of neutrophil-rich exudates. In summary, Duffy antigen expression is increased in the vascular beds and alveolar septa of the lung parenchyma during suppurative pneumonia, suggesting that Duffy antigen may have a functional role in the lung parenchyma during inflammation.     

Dimensions and alveolar surface of the ineralveolar septa of the rat lung]

The dimensions of the alveolar surface of interalveolar septa were studied in albino rats. The mean thickness of certain septa range from 3,1 to 6,2 mu, the mean thickness of the septa in different individuals being factually the same. A part of the alveolar surface of the septa represents the air-haematic barrier which occupies 51-64% of the total alveolar surface in different rats. The mean values of all the parameters in the animals studied are very close. It shows that the interalveolar septa are standard structures in their organization and dimensions both in the same animal and in different animals of the same species.     

Derepression of hepatocyte proliferation with changes in the functional state of the reticuloendothelial system

Experiments on rats and mice were performed to study the effects of different substances modifying RES functions on hepatocyte proliferation. It was shown that as early as 24 hours after Kupffer cell (KC) overloading with colloidal iron particles the number of hepatocytes in mitosis increased. The mitotic rate increased by 32 h and decreased between 48 and 72 h following iron injection. Forty-eight h after injection of latex particles the hepatocyte mitotic peak could be identified. Twenty-four and 48 h after zymozan injection DNA synthesis in sinusoidal liver cells correspondingly increased. Hepatocytes in mitosis appeared 5 days later, reaching the peak value after 9 days followed by a decrease 12 days after zymozan injection. The depression of the hepatocyte mitotic rate was also observed 9 days after BCG and 15 days after prodigiozan injection. The data are suggestive of the importance of KC as potential inducers of hepatocyte proliferation.     

GM-CSF mediates alveolar epithelial type II cell changes, but not emphysema-like pathology, in SP-D-deficient mice

Surfactant protein D (SP-D) is a member of the collectin subfamily of C-type lectins, pattern recognition proteins participating in the innate immune response. Gene-targeted mice deficient in SP-D develop abnormalities in surfactant homeostasis, hyperplasia of alveolar epithelial type II cells, and emphysema-like pathology. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is required for terminal differentiation and subsequent activation of alveolar macrophages, including the expression of matrix metalloproteinases and reactive oxygen species, factors thought to contribute to lung remodeling. Type II cells also express the GM-CSF receptor. Thus we hypothesized GM-CSF might mediate some or all of the cellular and structural abnormalities in the lungs of SP-D-deficient mice. To test this, SP-D (D-G+) and GM-CSF (D+G-) single knockout mice as well as double knockout mice deficient for both SP-D and GM-CSF (D-G-) were analyzed by design-based stereology. Compared with wild type, D-G+ as well as D+G- mice showed decreased alveolar numbers, increased alveolar sizes, and decreased alveolar epithelial surface areas. These emphysema-like changes were present to a greater extent in D-G- mice. D-G+ mice developed type II cell hyperplasia and hypertrophy with increased intracellular surfactant pools, whereas D+G- mice had smaller type II cells with decreased intracellular surfactant pools. In contrast to the emphysematous changes, the type II cell alterations were mostly corrected in D-G- mice. These results indicate that GM-CSF-dependent macrophage activity is not necessary for emphysema development in SP-D-deficient mice, but that type II cell metabolism and proliferation are, either directly or indirectly, regulated by GM-CSF in this model.     

Epidermal fatty acid-binding protein is increased in rat lungs following in vivo treatment with keratinocyte growth factor

Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

A Highlights from MBoC Selection: Mitotic regulation of fungal cell-to-cell connectivity through septal pores involves the NIMA kinase

Intercellular bridges are a conserved feature of multicellular organisms. In multicellular fungi, cells are connected directly via intercellular bridges called septal pores. Using Aspergillus nidulans, we demonstrate for the first time that septal pores are regulated to be opened during interphase but closed during mitosis. Septal pore–associated proteins display dynamic cell cycle–regulated locations at mature septa. Of importance, the mitotic NIMA kinase locates to forming septa and surprisingly then remains at septa throughout interphase. However, during mitosis, when NIMA transiently locates to nuclei to promote mitosis, its levels at septa drop. A model is proposed in which NIMA helps keep septal pores open during interphase and then closed when it is removed from them during mitosis. In support of this hypothesis, NIMA inactivation is shown to promote interphase septal pore closing. Because NIMA triggers nuclear pore complex opening during mitosis, our findings suggest that common cell cycle regulatory mechanisms might control septal pores and nuclear pores such that they are opened and closed out of phase to each other during cell cycle progression. The study provides insights into how and why cytoplasmically connected Aspergillus cells maintain mitotic autonomy.     

Isoprenaline-induced cell proliferation in mouse salivary glands: the effect of castration

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.