Increased serum ADAM8 concentration in patients with drug-induced eosinophilic pneumonia-ADAM8 expression depends on a the allergen route of entry.

BACKGROUND: ADAM8 (a disintegrin and a metalloprotease 8) has been linked to asthma and eosinophilic pneumonia (EP). ADAM8 cleaves a variety of substrates and is a sheddase for CD23, the low affinity IgE receptor. The concentration of soluble ADAM8 (sADAM8) is increased in bronchoalveolar lavage fluid (BALF) from patients with smoking-induced acute eosinophilic pneumonia (AEP) and chronic eosinophilic pneumonia (CEP), but not drug-induced EP (Drug-EP). In AEP, the BALF sADAM8 concentration significantly correlates with the soluble CD23 concentration (sCD23). METHODS: To evaluate the involvement of ADAM8 in the pathogenesis of eosinophilic pneumonia, we measured the concentrations of sADAM8 and its substrate, soluble CD23 (sCD23), in serum from patients with AEP, CEP, and Drug-EP. We also measured the change in the sADAM8 concentration after a provocation test. RESULTS: In contrast to the BALF findings, serum sADAM8 concentrations were increased in Drug-EP (mean+/-SEM; 639.6+/-49.15) and serum ADAM8 levels correlated positively with the serum sCD23 levels in patients with Drug-EP (P=0.0080, R(2)=0.8465). Serum sADAM8 concentrations were also increased in AEP (409+/-76.91) and CEP (644.7+/-87.03). Serum ADAM8 concentrations were also elevated after the provocation test. CONCLUSION: Serum ADAM8 concentrations were elevated in Drug-EP, although the sADAM8 concentrations were not increased in the BALF in Drug-EP. Thus, the pathogenesis of AEP and Drug-EP may be distinct with regard to allergen exposure; AEP may be caused by the inhalation of antigens, whereas Drug-EP may be caused by bloodstream antigens. These findings indicate that ADAM8 levels reflect the route of eosinophilic inflammation in EP.

Age and gender specific prevalence of HTLV-1.

BACKGROUND: The seroprevalence of Human T-cell Leukemia Virus Type 1 (HTLV-1) is female predominant despite the higher incidence of Adult T-cell Leukemia (ATL) in males. If the mother-to-child transmission of HTLV-1 is more common for male infants than in female infants, longer exposure to the virus for males may explain the paradoxically higher incidence of ATL. OBJECTIVES: To test the hypothesis that the seroprevalence of HTLV-1 is male predominant during adolescence. STUDY DESIGN: The presence of HTLV-1 antibody in 272,043 blood samples donated to a regional blood bank in an HTLV-1 high-endemic region was assessed. RESULTS: The entire population of female donors had a significantly higher seroprevalence compared to males (2.05% and 1.80%, respectively, p<0.0001). However, compared with male donors, the carrier rate for female donors was lower for the youngest subgroup (16-19 years, p=0.0011); was similar for the next two age subgroups (20-29 years and 30-39 years); and was significantly higher for the last two age subgroups (40-49 years and over 50-64 years, both p<0.0001). In general, older age subgroups led to higher seroprevalence in both genders. CONCLUSIONS: HTLV-1 infection is more common for males until after age 20, when male to female sexual transmission becomes likely. This suggests that mother-to-child transmission is more common for males.

Preclinical efficacy and safety of 1-deoxygalactonojirimycin in mice for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of alpha-galactosidase A (alpha-Gal A) activity. It has been shown that protein misfolding is primarily responsible for the enzyme deficiency in a large proportion of mutations identified in Fabry patients with residual enzyme activity, and 1-deoxygalactonojirimycin (DGJ) can effectively increase the residual enzyme activity in cultured patient's cells. Herein, we demonstrate the preclinical efficacy and safety of DGJ in transgenic mice that express human mutant alpha-Gal A activity. alpha-Gal A activity in heart, kidney, spleen, and liver was increased dose- and time-dependently. The mutant alpha-Gal A was increased in cardiomyocytes and distal convoluted tubules of the transgenic mice in a null background after 2 weeks of DGJ treatment. Globotriaosylceramide storage was remarkably reduced in kidney of mice after a 4-week treatment at a dosage of approximately 3 mg/kg body weight/day. The half-life of DGJ was less than 1 day in all major issues and that of the enzyme synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at approximately 30 mg/kg body weight/day for 9 weeks. Furthermore, no change was observed in appearance, growth, fertility, and life span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and is well tolerated in mice.

Elevated soluble ADAM8 in bronchoalveolar lavage fluid in patients with eosinophilic pneumonia.

BACKGROUND: ADAM (a disintegrin and metalloprotease) family members, characterized by a metalloprotease and a disintegrin domain, are membrane-anchored glycoproteins involved in proteolysis and cell adhesion. ADAM8 might have an important role in allergic inflammation. It can cleave a variety of substrates and is a sheddase for VCAM-1 and CD23, the low-affinity IgE receptors. METHODS: To evaluate the contribution of ADAM8 to the pathogenesis of eosinophilic pneumonia (EP), we measured the concentrations of soluble ADAM8 (sADAM8) and its substrates, soluble VCAM-1 (sVCAM-1) and soluble CD23 (sCD23), in bronchoalveolar lavage fluid from patients with smoking-induced acute eosinophilic pneumonia (AEP), chronic idiopathic eosinophilic pneumonia (CEP), and drug-induced eosinophilic pneumonia (drug-EP). RESULTS: The sADAM8 and sVCAM-1 concentrations were increased in AEP and CEP. The sCD23 concentration was elevated in AEP. In AEP, but not CEP, the sADAM8 concentration significantly correlated with those of both sVCAM and sCD23. CONCLUSION: The pathogenesis of AEP, CEP, and drug-EP was distinct with regard to ADAM8. Our results are the first to associate ADAM8 with eosinophilic responses and lung inflammation in humans.

Role of ADAM8 in experimental asthma.

A disintegrin and metalloprotease (ADAM) family members, characterized by a metalloprotease and a disintegrin domain, are membrane-anchored glycoproteins involved in proteolysis and cell adhesion. ADAM8 is specifically induced in the experimental murine asthmatic lung. To evaluate novel pathways involved in asthma pathogenesis, using ADAM8 transgenic mice (ATMS2) in a murine model of asthma. Massive cellular infiltrates in peribronchovascular and interstitial lesions were observed in control mice, while in ATMS2 mice there were only occasional. Vascular cell adhesion molecule (VCAM-1) is involved in specific eosinophil adhesions via alpha4beta1 integrin. VCAM-1 shedding was mediated by the ADAM8 metalloprotease. Endothelial cell shedding of VCAM-1 was increased in ATMS2-stimulated human umbilical endothelial cells. ADAM8-mediated shedding of VCAM-1 might be important for the suppression of experimental asthma. Our data suggest that ADAM8 is a useful therapeutic target.

Involvement of CD14 in lipopolysaccharide- induced liver injury in mice pretreated with Propionibacterium acnes.

OBJECTIVE: The aim of this study was to elucidate the role of CD14 in the Propionibacterium acnes-lipopolysaccharide (LPS) system. METHODS AND RESULTS: CD14 transgenic mice (M14M), which expressed heterotopic CD14 and showed decreased responses to LPS in vivo, were used. Seven days after priming, the size of granulomas induced by an intraperitoneal administration of P. acnes in the M14M mice was smaller than that in the nontransgenic mice. The number of CD14-positive cells in granulomas was also decreased in the M14M mice compared to the nontransgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in the nontransgenic mice but not in the M14M mice. Seven days after priming, tumor necrosis factor-alpha expression was found in monocytic cells in granulomas and Kupffer cells in the nontransgenic mice and was significantly upregulated after LPS injection, whereas the expression was very weak in these cells in the M14M mice. CONCLUSIONS: CD14 plays a role in the P. acnes-LPS system in both priming and induction phases.

Expression of splice variants of the human ADAM15 gene and strong interaction between the cytoplasmic domain of one variant and Src family proteins Lck and Hck.

OBJECTIVE: The aim of the present study was to show variant species of ADAM15 and unique Src homology 3 (SH3)-binding motifs, which strongly bound Src family proteins compared with ADAM15. METHODS AND RESULTS: RT-PCR using primers for the cytoplasmic domain revealed the presence of different species, designated ADAM15v1 and ADAM15v2, which had characteristic SH3-binding class I and class II motifs. The mRNA of ADAM15v1 and ADAM15v2 was mainly found in peripheral blood mononuclear cells, T lymphocytes and monocytic cell lines. ADAM15v2 protein interacted more strongly with the Src family proteins Lck and Hck than did ADAM15 protein, as examined by pull-down analysis and immunoprecipitation followed by immunoblot analysis. The binding with Lck and Hck was enhanced by the phosphorylation of ADAM15v2 protein. CONCLUSIONS: These results suggest that the cytoplasmic domain of ADAM15v2 strongly interacts with Lck and Hck and regulates leukocyte function.

Roles of CD14 in LPS-induced liver injury and lethality in mice pretreated with Propionibacterium acnes.

The mechanism of the liver damage and lethality in Propionibacterium acnes (P. acnes)-LPS system remains obscure. To examine the role of CD14 in the system, M14M mice, in which CD14 was expressed heterotopically under the control of the metallothionein promoter were used. The production of soluble CD14 (sCD14) was increased by both P. acnes - priming and LPS challenge (1 microg per mouse) in both nontransgenic and M14M mice, although the plasma level was much higher in M14M nontransgenic than mice. The size of granulomas induced by an intraperitoneal administration of P. acnes in M14M mice 7 days after priming was smaller than that in nontransgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in nontransgenic mice but not in M14M mice. The challenge dose resulted in almost 90% lethality in nontransgenic mice but not in M14M mice 24h after challenge. TNF-alpha, IFN-gamma, IL-12, IL-18 and inducible nitric oxide synthase (iNOS) mRNA expressions produced by LPS challenge in M14M mice were low compared with those in nontransgenic mice. IL-18 mRNA expression was upregulated in P. acnes-primed nontransgenic mice but not in M14M mice. These results suggest that the high sCD14 concentration may account for less marked liver damage in M14M mice. Increase in the challenge dose of LPS (2 microg per mouse) resulted in increased lethality of M14M mice without liver damage. The levels of endothelial cell leukocyte adhesion molecule (ELAM)-1 mRNA expression in several organs in M14M mice 1-3h after LPS challenge were, however, lower than those in nontransgenic mice. The high sCD14 concentration may stimulate endothelial cell activation, which may account for lethality without liver damage in M14M mice. Thus, CD14 is involved in both the priming and induction phases as well as lethality in P. acnes-LPS system.

Structure and expression of the murine ADAM 15 gene and its splice variants, and difference of interaction between their cytoplasmic domains and Src family proteins.

The murine cell surface antigen ADAM 15 is a transmembrane glycoprotein that is expressed in a variety of cells including monocytic and T cell lines and consists of a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, and an epidermal growth factor (EGF)-like domain in the extracellular region. The cytoplasmic domain comprises 103 amino acids containing proline-rich endophilin I, Src homology 3 (SH3), and phox homology domain-containing protein (SH3PX1) binding motifs. The ADAM15 gene is composed of 21 exons and 20 introns and spans approximately 10 kb. The transcription initiation site of the ADAM15 gene was defined by an oligonucleotide-capping method. Reverse transcription (RT)-PCR using primers of the cytoplasmic domain of ADAM15 revealed the presence of different ADAM15 species designated ADAM15v1 and ADAM15v2, respectively, that had characteristic SH3-binding class I and/or class II motifs. The ADAM15v1 and ADAM15v2 genes consist of an extra one exon and two exons, respectively, which exist in intron 19 of the ADAM15 gene. The expression of ADAM15v1 and ADAM15v2 mRNA was found in T lymphocyte and monocyte lines. ADAM15v2 protein interacted more strongly with the Src family proteins Lck and Src than ADAM15 protein, when examined by pull-down and immunoprecipitation followed by immunoblot analysis using a T lymphocyte line. Phosphorylation of ADAM15v2 protein markedly enhanced the binding with Lck. These results suggest that the cytoplasmic domain of ADAM15v2 strongly interacts with Lck and plays an important role in T lymphocytes.

Expression of murine N-MYC by insertion of retrovirus sequences in a murine macrophage cell line (RAW264).

RAW264 cells, reported to be originated from Abelson-virus-induced B lymphomas, are widely used as a murine monocyte cell line. We found that RAW264 show enhanced expression of murine N-MYC. Murine cDNA clones associated with N-MYC were separated from (lambda)gt11 cDNA library constructed by using mRNA from the macrophage cell line, RAW264 cells. Sequencing analysis of the longest cDNA clone N-MYCL showed that the length of the coding region was 18 bases shorter than that of the predicted full length N-MYC cDNA, and the 3' untranslated region had the 5' long terminal repeat (LTR) sequence of the Moloney-like proviral sequence, suggesting the expression of N-MYC by insertion of the proviral sequence. This suggests that expression of N-MYC plays a role in the establishment of macrophage cell line RAW264. Integration of LTR and overexpression of the N-MYC gene might have existed in the parental lymphoma cells, playing a role in the development of lymphoma or in the establishment of macrophage cell line.