Successful Steroid Therapy for Lipoid Pneumonia Developing After Allogeneic Hematopoietic Stem Cell Transplant: A Case Report.

Lipoid pneumonia is an uncommon noninfectious inflammatory lung disease characterized by lipid deposition in the alveoli, and its etiology and treatment have not been elucidated. We report the case of a 32-year-old woman who developed lipoid pneumonia 9 months after allogeneic hematopoietic stem cell transplant for chronic myelogenous leukemia in lymphoid blast crisis. She complained of progressive cough and dyspnea shortly after discontinuation of immunosuppressive therapy given for graft-vs-host disease. Computed tomography demonstrated diffuse ground-glass opacities in the lungs, and pulmonary function test revealed restrictive impairment. Bronchoalveolar lavage fluid showed milky appearance, and transbronchial lung biopsy specimen revealed foamy macrophages infiltrating the alveoli. Based on these findings, she was diagnosed as having lipoid pneumonia. Prednisolone (1 mg/kg/d) promptly improved the symptoms, pulmonary shadows, and pulmonary function. The findings and clinical course of this case suggest that lipoid pneumonia should be recognized as one of the pulmonary complications of allogeneic hematopoietic stem cell transplantation.

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis and in vitro cytokine-stimulated blood eosinophils.

Investigation of differentially expressed genes in eosinophils of patients with allergic diseases such as atopic dermatitis (AD) will provide important information for elucidating possible mechanisms of pathology. To identify novel genes that are expressed in AD, we compared gene expression in samples of peripheral blood eosinophils from AD patients and healthy volunteers. RNA was extracted from peripheral blood eosinophils. The expression of various genes, such as those for cytokine receptors, eosinophil activation marker, platelet activating factor (PAF) receptor, eosinophil-specific granular proteins and apoptosis-related genes, was confirmed using real-time reverse transcription-polymerase chain reaction (RT-PCR). Peripheral blood eosinophils of healthy volunteers were also isolated and stimulated for introduction of various cytokines. RNA was extracted and gene expression was monitored. Several genes, such as those for cytokine receptors (granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha and beta chain and interleukin (IL)-3 receptor alpha chain), CD44 and PAF receptor were expressed at significantly higher levels in AD patients than in healthy volunteers. In addition, the anti-apoptotic genes, bcl-2 and bcl-xL, were expressed at increased levels in AD patients. No single gene expression correlated with clinical markers, such as eosinophil count or IgE levels. Expression of GM-CSF receptor beta chain and IL-3 receptor alpha chain in isolated blood eosinophils of healthy volunteers was stimulated by IL-5, IL-4, interferon (IFN)-gamma and GM-CSF. Expression of bcl-2 and bcl-xL was also increased after stimulation with IL-5, IL-4 or IFN-gamma. The in vitro enhancement of cytokine-stimulated gene expression correlated well with the enhancement observed in clinical samples of eosinophils, suggesting that cytokines may affect gene expression in vivo in eosinophils of patients with AD.

Gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells.

Mast cells (MCs) and eosinophils are thought to play important roles in evoking allergic inflammation. Cell-type--specific gene expression was screened among 12,000 genes in human MCs and eosinophils with the use of high-density oligonucleotide probe arrays. In comparison with other leukocytes, MCs expressed 140 cell-type--specific transcripts, whereas eosinophils expressed only 34. Among the transcripts for expected MC-specific proteins such as tryptase, major basic protein (MBP), which had been thought to be eosinophil specific, was ranked fourth in terms of amounts of increased MC-specific messenger RNA. Mature eosinophils were almost lacking this transcript. MCs obtained from 4 different sources (ie, lung, skin, adult peripheral blood progenitor--derived and cord blood progenitor--derived MCs, and eosinophils) were found to have high protein levels of MBP in their granules with the use of flow cytometric and confocal laser scanning microscopic analyses. The present finding that MCs can produce abundant MBP is crucial because many reports regarding allergic pathogenesis have been based on earlier findings that MBP was almost unique to eosinophils and not produced by MCs. (Blood. 2001;98:1127-1134)

Gene expression accompanied by differentiation of cord blood-derived CD34+ cells to eosinophils.

To clarify the relation between the expression of genes such as eosinophil-specific granular proteins and cytokine receptors and the pathogenesis of allergic disease, cord blood-derived CD34+ cells were cultured and differentiated into eosinophils. Gene expression in the cells during the differentiation was determined by real-time reverse transcription PCR (ABI PRISM 7700). CD34+ mononuclear cells cultured with stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and IL-5 in Iscove's MEM, and proliferated until the 2nd week, when the cell number reached a plateau. Under these conditions, more than 90% of the cells differentiate into mature eosinophils in 3 weeks. The expression of major basic protein and eosinophil-derived neurotoxin in the treated cells increased until week 2 and decreased between week 2 and 3. However, the expression of membrane receptor genes, such as IL-5 receptor (alpha chain), IL-3 receptor (alpha chain), GM-CSF-alpha receptor, GM-CSF-beta receptor, CC chemokine receptor 3, interferon-gamma receptor, platelet-activating factor receptor and leukotriene D4 receptor, increased until the 3rd week of eosinophil maturation. Our study suggests that the in vitro eosinophil differentiation and maturation model is useful for clarifying the relation between eosinophil-specific gene expression during allergic diseases and the progression of the disease.

A factor derived from polymorphonuclear leukocytes enhances interleukin-1-induced synovial cell collagenase and prostaglandin E2 production in rats.

We found that short-term culture medium and homogenate of casein-induced rat peritoneal polymorphonuclear leukocytes (PMN) markedly induced collagenase and prostaglandin E2 (PGE2) production by normal rat synovial cells and these effects were abrogated by anti-(rat interleukin-1 alpha) (IL-1 alpha) polyclonal antibodies. However, collagenase activity and PGE2 induced by recombinant rat IL-1 alpha were less than those induced by rat PMN culture medium. It was also proved by radioimmunoassay that rat PMN culture medium contains a relatively small amount of IL-1 alpha. The introduction of IL-1 alpha-deleted PMN culture medium and recombinant rat IL-1 alpha together into the synovial cell culture system revealed that IL-1 alpha deleted PMN culture medium has a significant enhancing activity on IL-1 alpha-induced synovial cell collagenase and PGE2 production. This new factor, which was shown to be a negatively charged protein of about 80 kDa, may have important roles in connective tissue destruction and chronic inflammation in diseases such as rheumatoid arthritis.

Heterogeneity in lysosomal fusion with phagocytic vesicles and cell membrane in non-phagocytosing guinea-pig polymorphonuclear leukocyte.

The fusion of lysosomes and other granules with phagocytic vesicles (endo-fusion) and cell membrane (exocytosis) was simultaneously examined in non-phagocytosing guinea-pig polymorphonuclear leukocytes (PMNs). beta-Glucuronidase as a typical lysosomal enzyme, acid phosphatase as another lysosomal enzyme, and alkaline phosphatase as a specific granule enzyme were assayed. PMNs released these three enzymes in the presence of serum but the extents of exocytosis differed considerably: release of beta-glucuronidase, alkaline phosphatase and acid phosphatase was 6.9, 4.3 and 3.3%, respectively. Acid phosphatase was released even in the absence of serum, whereas the other two enzymes were not. These three enzymes showed also different responses in endofusion: the preformed phagocytic vesicles fused with the granules containing beta-glucuronidase and acid phosphatase, but scarcely fused with those containing alkaline phosphatase, although all these enzymes were recovered in increasing amounts in the phagolysosome fraction when the cells were allowed to phagocytose continuously. These results suggest that fusion of these three types of granules with phagocytic vesicles (endo-fusion) and plasma membrane (exocytosis) is heterogeneous and regulated by different mechanisms in guinea-pig PMNs.

Effect of PGI2 on platelet binding to partially denuded endothelial monolayer in vitro.

We have developed a new model for the investigation of platelet interaction with injured vascular endothelium. This involves the quantitative detection of platelet binding to a partially denuded endothelial cell monolayer in vitro. Porcine arterial endothelial monolayer, cultured on collagen gel containing fibrinogen and fibronectin, was partially denuded and the binding of 51Cr-platelets was measured. A synergistic increase in platelet binding was observed in the presence of fibrinogen and fibronectin. A distinct aggregation of platelets along the edge of the denuded area of the endothelial monolayer was seen. Prostacyclin (PGI2) inhibited platelet aggregation, although adhesive platelets were still present at denuded sites.

Collagenase production in the exudate of a novel adjuvant-induced air pouch inflammation model in rats.

To elucidate the role of collagenase in tissue destruction in chronic inflammation (e.g., in rheumatoid arthritis), we established a novel air pouch model in rats. Ten ml of air was injected subcutaneously on the dorsa of F344 rats, and 24 hours thereafter, Freund's complete adjuvant was injected into the air pouch. A remarkably high activity of trypsin-activated collagenase was detected dose-dependently in the pouch fluid at 8 days after the challenge injection. Concomitantly with the increase of collagenase activity, the weight of granuloma formed in the pouch decreased, suggesting that collagenase participates in the resorption of granuloma tissues.

The inhibitory effect of a novel antiatheromatous agent, E5050, on the intimal thickening of aorta in cholesterol-fed rabbit in vivo.

The development of atheromatous lesions in the aortic arch of 0.5% cholesterol-fed rabbits was biochemically and morphologically examined. The animals were killed at week twelve (Cont-12W) or sixteen (Cont-16W). Both the micrographic and biochemical studies showed that the main atheromatous lesions in the Cont-12W group were fatty streaks, whereas those in the Cont-16W group were fibrous plaques. In these models, oral ingestion of 0.2% and 0.4% E5050, which has an antiproliferative effect on smooth muscle cells, had no effect on the surface involvement or the lipid content of the aortic arch at the sixteenth week, but reduced the degree of intimal thickening and the DNA content in the aortic arch in a dose-dependent manner. These results strongly suggest that E5050 suppresses the intimal thickening through its inhibitory effect on the proliferation of smooth muscle cells.

Enzymatic hydrolysis of indometacin farnesil, a prodrug of indomethacin, by carboxylesterase in cultured synovial cells.

The hydrolysis of indometacin farnesil (IMF) in the synovial cells of rat and human and the subcellular fractions of rat liver were investigated in relation to the inhibition of prostaglandin E2 (PGE2) production in the synovial cells. The inhibition of PGE2 production in cultured human synovial cells by anti-inflammatory drugs was potent in the order of IND, IMF and acetyl salicylic acid. However, when the cells were pretreated with IMF, the inhibitory activity of IMF was retained even after the compound was washed out from the medium. No duration of the inhibition was seen in the pretreatment of the cells with IND or acetyl salicylic acid. These results suggest that IMF incorporated into the synovial cells was hydrolyzed gradually to IND. In fact, IMF was taken up by rat synovial cells in culture and considerable amount of IND, which increased with culture period, was found out in the cells. Furthermore, the IMF hydrolase activity was found in microsomal and lysosomal fractions of rat liver, and the hydrolase was identified as carboxylesterase by using bis-(p-nitrophenyl) phosphate, a specific inhibitor of carboxylesterase.

Antiinflammatory properties of E5090, a novel orally active inhibitor of IL-1 generation.

E5090 is a novel orally active inhibitor of IL-1 generation without cyclooxygenase-inhibiting activity. The effects of E5090 on several inflammatory animal models were investigated in rats. In adjuvant arthritis, E5090 suppressed both the paw swelling and the enhancements of ESR and number of peripheral blood leucocytes, like the steroidal antiinflammatory drug prednisolone. However, the thymus was not withered by E5090 though it was by prednisolone. In type II collagen-induced arthritis, E5090 inhibited paw swelling and joint destruction. E5090 was effective in acute inflammatory models such as carrageenin-induced paw edema, and adjuvant-induced local hyperthermia, and also showed analgesic effects against inflammatory pain and antipyretic effects. The results suggest that this orally active inhibitor of IL-1 generation, E5090, may be a therapeutically useful antiinflammatory drug with a novel mechanism of action.

A novel inhibitor of IL-1 generation, E5090: in vitro inhibitory effects on the generation of IL-1 by human monocytes.

E5090 is an orally active inhibitor of IL-1 generation, being converted in vivo into the pharmacologically active deacetylated form (DA-E5090). In vitro effects of DA-E5090 on the generation of IL-1 by human monocytes stimulated with LPS were examined. DA-E5090 inhibited both IL-1 alpha and IL-1 beta generation by human monocytes stimulated with 1 microgram/ml of LPS in a dose dependent-manner (1-10 microM), as determined by LAF assay and ELISA. Northern blotting analysis indicated that DA-E5090 inhibits transcription of IL-1 alpha and IL-1 beta m-RNAs.

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes.

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studied in vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 micrograms/ml) in a dose-dependent manner (1-10 microM). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC50:0.084 microM), aurothioglucose (11.5 microM) and lobenzarit (75.0 microM), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 microM). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.

Effect of PGI2 on transcellular transport of fluorescein dextran through an arterial endothelial monolayer.

The effects of prostacyclin (PGI2) and stable derivatives of PGI2, such as isocarbacyclin (PGI2 deriv. (A] and isocarbacyclin methyl ester (PGI2 deriv. (B)), on junctional transport of fluorescein dextran (FD) through cultured porcine arterial endothelial cells were investigated. These PGI2S inhibited the transcellular transport dose-dependently. After the elimination of PGI2, its inhibitory effect persisted for at least 1 hr. A good correlation was found between increase of cAMP and the potency of inhibition. Increase of cAMP after PGI2 treatment seemed to be involved in the inhibition of FD transport.

Transcellular transport of fluorescein dextran through an arterial endothelial cell monolayer.

Transcellular transport of fluorescein dextran (FD) of various molecular weights (4K, 10K, 20K, 70K and 150K daltons) through porcine arterial endothelial cells cultured on a type I collagen gel supported by a dacron sheet was studied and compared with the transport of low density lipoprotein labeled with rhodamine B (RB-LDL) described previously (Hashida et al., Cell Struct. Funct. 11, 31-42, 1986). The rate of FD transport through the monolayer depended on the size of the FD. FD transport was not temperature-dependent and was not a saturable process. Our findings show that FD transport differs from RB-LDL transport which is temperature- and dose-dependent. The mechanism of the transport of FD is compared with that of RB-LDL.

Transcellular transport of lipoprotein through arterial endothelial cells in monolayer culture.

To study the mechanism of lipoprotein transport through arterial endothelial cells, porcine endothelial cells were cultured on gelated type I collagen supported by a dacron sheet, and the transport of low density lipoprotein (LDL) labeled with rhodamine B isothiocyanate (RB-LDL) through the cells was measured. Light and scanning electron microscopy showed that the cells on the gel were confluent. There was little RB-LDL transport through the endothelial monolayer at 0 degrees C. RB-LDL transport through the monolayer at 37 degrees C was dose-dependent saturable at 0.4 mg protein/ml. The transport was energy-dependent, since its rate was affected by temperature and was inhibited by a combination of 2-deoxyglucose (50 mM) and NaN3 (10 mM). RB-LDL was shown not to be degraded during transport.

Stimulation of prostaglandin synthesis in cultured rat synovial cells by a factor derived from polymorphonuclear leukocytes.

Stimulation of synovial cell prostaglandin production by a factor obtained from casein-induced peritoneal polymorphonuclear (PMN) cells has been investigated. Both the extract and short time cultured medium of rat peritoneal PMN cells stimulate prostaglandin (PG)E2 production as well as collagenase production in the culture of rat synovial cells. PGE2 production by the cells in the presence of the PMN factor is much faster (5 to 24 hr) than collagenase production (24 hr or later, Biomedical Res. 3, 506-516, 1982). This stimulating factor is confirmed to be derived from PMN cells, based on the purification of the cells from peritoneal exudate cells by the Ficoll-Urographin method. Elution profile of the factor on gel filtration has indicated that both PGE2 and collagenase productions by synovial cells are stimulated by the same effluent fractions corresponding to molecular weights of 15,000 - 20,000 daltons and 30,000 - 40,000 daltons. These results suggest that PMN cells are involved in PG production as well as collagenase production in the inflamed tissue by stimulating connective tissue cells such as synovial cells.