Publications

Aspergillus fumigatus (AF) is a ubiquitous mold and the most common cause of invasive aspergillosis (IA) in immunocompromised patients. In stem cell transplant recipients, IA now occurs most frequently in the setting of therapy with corticosteroids, including methylprednisolone (MP). We showed previously that gliotoxin (GT), an AF-derived mycotoxin, induces apoptosis in monocytes and dendritic cells, resulting in the suppression of AF-specific T cell responses. We examined the ability of GT to induce apoptosis in polymorphonuclear leukocytes (PMN) and assessed GT effects on important neutrophil functions, including phagocytic function, degranulation, myeloperoxidase activity, and the production of reactive oxygen species (ROS). In contrast to its effects on monocytes, PMN remained resistant to GT-mediated apoptosis. Although many essential neutrophil functions were unaffected, GT inhibited phagocytosis and also induced a decrease in ROS generation by PMN. In contrast, MP therapy potentiated ROS production, suggesting a mechanism that may facilitate tissue injury in IA. Distinct from its effects on untreated PMN, GT augmented ROS production in MP-treated PMN. Our results suggest that although GT may suppress the adaptive immune response, GT may also serve to increase PMN-mediated inflammation, which is likely to play an important role in tissue destruction in the setting of IA.

The integrin receptor alphavbeta5 controls two independent forms of interactions of the retinal pigment epithelium (RPE) with adjacent photoreceptor outer segments that are essential for vision. Alphavbeta5 localizes specifically to apical microvilli of the RPE and contributes to retinal adhesion that maintains RPE contacts with intact outer segments at all times. Additionally, alphavbeta5 synchronizes diurnal bursts of RPE phagocytosis that clear photoreceptor outer segment fragments (POS) shed in a circadian rhythm. Dependence of retinal phagocytosis and adhesion on alphavbeta5 receptors suggests that the extracellular matrix ensheathing RPE microvilli contains ligands for this integrin. Here we studied mice lacking expression of functional MFG-E8 to test the contribution of this integrin ligand to alphavbeta5 functions in the retina. Lack of MFG-E8 only minimally reduced retinal adhesion. In contrast, lack of MFG-E8, like lack of alphavbeta5 receptor, eliminated alphavbeta5 downstream signaling involving the engulfment receptor MerTK and peak POS phagocytosis, both of which follow light onset in wild-type retina. MFG-E8-deficient RPE in primary culture retained normal epithelial morphology and levels of apical alphavbeta5 receptors, but showed impaired binding and engulfment of isolated POS. Soluble or POS-bound recombinant MFG-E8 was sufficient to fully restore phagocytosis by MFG-E8-deficient RPE. Furthermore, MFG-E8 supplementation strongly increased POS binding by wild-type and MerTK-deficient RPE, but did not affect POS binding by RPE lacking alphavbeta5. Thus, MFG-E8 stimulates rhythmic POS phagocytosis by ligating apical alphavbeta5 receptors of the RPE. These results identify MFG-E8 as the first extracellular ligand in the retina that is essential for diurnal POS phagocytosis.

ATP-binding cassette (ABC) transporter, family 12 (ABCA12), a member of the ABC superfamily, facilitates the delivery of lipids to lamellar bodies (LB) in keratinocytes, which is critical for permeability barrier function. Recently, gene mutations of ABCA12 were found to underlie Harlequin ichthyosis and lamellar ichthyosis, two devastating skin disorders. Previously we and others have demonstrated that peroxisome proliferators-activated receptors (PPARs) and liver X receptor (LXR) activation improved epidermal permeability barrier homeostasis by stimulating keratinocyte differentiation, lipid synthesis, and increasing LB formation/secretion. Here we report that both PPAR-gamma and -beta/delta activators markedly stimulate ABCA12 mRNA expression in cultured human keratinocyte (CHK) in a dose- and time-dependent manner. Increased ABCA12 mRNA levels are accompanied by an increase in ABCA12 protein, suggesting biological importance of this upregulation. LXR activators also increase ABCA12 mRNA levels in CHK, but to a lesser extent. In contrast, activators of PPAR-alpha, RAR, RXR, or vitamin D receptor did not alter ABCA12 expression. Two major ABCA12 alternative transcripts and their corresponding proteins are also expressed and upregulated by PPAR or LXR activator in both undifferentiated and differentiated CHK. Together, our data demonstrate that PPAR and LXR activators increase ABCA12 expression, providing an additional mechanism by which PPAR and LXR activators promote epidermal permeability barrier homeostasis.

Extracellular calcium-sensing receptors (CaRs) and metabotropic or type B gamma-aminobutyric acid receptors (GABA-B-Rs), two closely related members of family C of the G protein-coupled receptor superfamily, dimerize in the formation of signaling and membrane-anchored receptor complexes. We tested whether CaRs and two GABA-B-R subunits (R1 and R2) are expressed in mouse growth plate chondrocytes (GPCs) by PCR and immunocytochemistry and whether interactions between these receptors influence the expression and function of the CaR and extracellular Ca(2+)-mediated cell differentiation. Both CaRs and the GABA-B-R1 and -R2 were expressed in the same zones of the growth plate and extensively colocalized in intracellular compartments and on the membranes of cultured GPCs. The GABA-B-R1 co-immunoprecipitated with the CaR, confirming a physical interaction between the two receptors in GPCs. In vitro knockout of GABA-B-R1 genes, using a Cre-lox recombination strategy, blunted the ability of high extracellular Ca(2+) concentration to activate phospholipase C and ERK1/2, suppressed cell proliferation, and enhanced apoptosis in cultured GPCs. In GPCs, in which the GABA-B-R1 was acutely knocked down, there was reduced expression of early chondrocyte markers, aggrecan and type II collagen, and increased expression of the late differentiation markers, type X collagen and osteopontin. These results support the idea that physical interactions between CaRs and GABA-B-R1s modulate the growth and differentiation of GPCs, potentially by altering the function of CaRs.