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Background Eosinophils are multifunctional granulocytes capable of releasing various cytokines, chemokines, and lipid mediators. We previously reported dysregulated fatty acid metabolism in peripheral blood-derived eosinophils from patients with severe asthma. However, functional characteristics of eosinophils present in allergic inflammatory tissues remain largely uncharacterized. Methods We established a method for isolating CD69hi CCR3low CXCR4- siglec-8int eosinophils from nasal polyps of patients with eosinophilic rhinosinusitis (NP-EOS). Multi-omics analysis including lipidomics, proteomics, and transcriptomics was performed to analyze NP-EOS as compared to peripheral blood-derived eosinophils from healthy subjects (PB-EOS). Results Lipidomic analysis revealed impaired synthesis of prostaglandins and 15-lipoxygenase (15-LOX)-derived mediators, and selective upregulation of leukotriene D4 production. Furthermore, proteomics and transcriptomics revealed changes in the expression of specific enzymes (GGT5, DPEP2, and 15-LOX) responsible for dysregulated lipid metabolism. Ingenuity pathway analysis indicated the importance of type 2 cytokines and pattern recognition receptor pathways. Stimulation of PB-EOS with eosinophil activators IL-5, GM-CSF, and agonists of TLR2 and NOD2 mimicked the observed changes in lipid metabolism. Conclusion Inflammatory tissue-derived eosinophils possess a specific phenotype with dysregulated fatty acid metabolism that may be targeted therapeutically to control eosinophilic inflammatory diseases.

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.

The membrane-bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase-negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single-chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two-chain enzyme with its specific inhibitor HAI-1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI-1 complexes. In cancer cell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor-free, two-chain active form. Trypsin seemed to activate the membrane-bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis.

The basement membrane protein laminin-5 promotes cell adhesion and migration. The carboxyl-terminal G3 domain in the alpha3 chain is essential for the unique activity of laminin-5. To investigate the function of the G3 domain, we prepared various recombinant laminin-5 forms with a partially deleted or mutated G3 domain. The deletion of the carboxyl-terminal 28 amino acids (region III) markedly decreased the cell adhesion activity with a slight loss of the cell motility activity toward BRL and EJ-1 cells. This change was attributed to the loss of Lys-Arg-Asp sequence. Further deletion of 83 amino acids (region II) led to almost complete loss of the cell motility activity. All charged amino acid residues tested in this region were not responsible for the activity loss. These results suggest that the G3 domain contains two distinct regions that differently regulate cell adhesion and migration. Analysis of laminin-5 receptors showed that integrins alpha3beta1, alpha6beta1, and alpha6beta4 had different but synergistic effects on cell adhesion and migration on laminin-5. However, the structural change of the G3 domain appeared not to change integrin specificity. The present study demonstrates that the G3 domain in laminin-5 plays a central role to produce different biological effects on cells.

Laminin-5 (alpha3beta3gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.