Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.     

Separation and mass spectrometric characterization of covalently bound skin ceramides using LC/APCI-MS and Nano-ESI-MS/MS

Ceramides covalently bound to keratinocytes are essential for the barrier function of the skin, which can be disturbed in diseases, such as psoriasis and atopic dermatitis. These ceramides of the classes omega-hydroxyacyl-sphingosine and omega-hydroxyacyl-6-hydroxysphingosine contain an omega-hydroxy fatty acid. For their separation and identification, a new analytical approach based on normal phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry and tandem nano-electrospray mass spectrometry, respectively, is presented here. Tandem mass spectrometry provided structural information about the sphingoid base as well as the fatty acid moieties. The chain lengths of the bases ranged from C12 to C22, the chain lengths of the fatty acids varied between C28 and C36. In total, 67 ceramide species have been identified in human skin. The analytical methods presented in this work can be helpful for investigating alterations in the ceramide composition of the skin as seen in psoriasis, atopic dermatitis, and diseases with impaired epidermal barrier function.     

Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding

The cornified lipid envelope (CLE) is a lipid monolayer covalently bound to the outside of corneocytes and is part of the stratum corneum (SC). The CLE is suggested to act as a scaffold for the unbound SC lipids. By profiling the bound CLE ceramides, a new subclass was discovered and identified as an omega-hydroxylated dihydrosphingosine (OdS) ceramide. Bound glucosylceramides were observed in superficial SC layers of healthy human skin. To investigate the relation between bound and unbound SC ceramides, the composition of both fractions was analyzed and compared. Selectivity in ceramide binding towards unsaturated ceramides and ceramides with a shorter chain length was observed. The selectivity in ceramide species bound to the cornified envelope is thought to have a physiological function in corneocyte flexibility. Next, it was examined if skin models exhibit an altered bound ceramide composition and if the composition was dependent on liver X-receptor (LXR) activation. The effects of an LXR agonist and antagonist on the bound ceramides composition of a full thickness model (FTM) were analyzed. In FTMs, a decreased amount of bound ceramides was observed compared to native human skin. Furthermore, FTMs had a bound ceramide fraction which consisted mostly of unsaturated and shorter ceramides. The LXR antagonist had a normalizing effect on the FTM bound ceramide composition. The agonist exhibited minimal effects. We show that ceramide binding is a selective process, yet, still is contingent on lipid synthesized.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Ceramide biosynthesis in keratinocyte and its role in skin function

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1–CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Characterization of glycosyl phosphatidylinositol lipids of parasitic protozoans: Leishmania mexicana mexicana promastigotes, Trypanosoma cruzi Peru epimastigotes and Tritrichomonas foetus

Glycosyl phosphatidylinositol lipids of cultured L.mex, mexicana LV732 promastigotes, T. cruzi Peru epimastigotes and Tritrichomonas foetus have been isolated and characterized using metabolic labelling and chromatographic and mass spectrometric (MS) techniques. TLC of the unsaponifiable lipid fractions of L. mex. mexicana and T. cruzi obtained from DEAE Sephadex A-25 followed by Iatrobead column chromatography showed three inositol phosphate-containing lipid components. [3H]myo-inositol, [3H]palmitic acid or H3 32PO4 lipid precursors were incorporated into these three lipid components. Fraction 2 (LM2 and TCP-2) comprises inositol phosphate ceramides. The other two fractions appear to contain mono-O-alkyl and di-O-alkyl glycerol inositol phosphates. Lyso-1-O-alkyl phosphatidylinositols could be cleaved by treatment of PI-specific phosphalipase C. The di-O-alkyl-phospho inositols of these parasites being the first dialkylglycerol lipids reported from eukaryotic membranes raises the possibility of chemotherapy for leishmaniasis and trypanosomiasis based upon functional impairment of alkyl ether lipids. Tritrichomonas foetus contains two major glycophosphosphingolipids, designated TF1 and TF2, which are metabolically labelled with [3H]myo-inositol and H3 32PO4. Both lipids contained ceramides. The major ceramide contains the 18:0 and 18:1 bases and 16:0 N-acyl group. The major glycolipid fraction (TF1) contains fucose linked to inositol diphosphate; one of the phosphates being linked to the ceramide moiety, and the other to ethanolamine. TF1 appears to be a novel class of glycophosphosphingolipid, which may be a part of a membrane anchor.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Rapid transbilayer movement of ceramides in phospholipid vesicles and in human erythrocytes

The transbilayer diffusion of unlabeled ceramides with different acyl chains (C6-Cer, C10-Cer, and C16-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (approximately 0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 degrees C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after approximately 15 min of incubation at 20 degrees C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography

This study reports a single-step analysis of the molecular species of endogenous ceramides and of the ceramide moiety of sphingomyelins in biological samples, using gas liquid chromatography (GLC). Silylated sphingomyelins were quantitatively converted to monosilylated ceramide upon injection into GLC, whereas the free ceramides were di-silylated on the primary and secondary alcohol function, as confirmed by mass spectrometry. The reproducible shift of the retention times between the mono- and di-silylated derivatives enables simultaneous quantification of the variety of sphingomyelin and ceramide molecular species. Overlapping diacylglycerols were first removed by a mild alkaline treatment of the lipid extract. The lowest detection limit (5 pmol) did not allow for identification of free ceramides in human plasma, but 17 molecular species of ceramides derived from sphingomyelins were quantified, from NC16:0 up to NC24:1. By contrast, three major free ceramides (NC16:0, NC24:0, and NC24:1) were quantified in HEPG2 and Chinese hamster ovary (CHO) cells. Upon induction of apoptosis in CHO cells by C6-ceramide, we could follow the disappearance of the C6-ceramide, its partial conversion to C6-sphingomyelin, and the prominent increase of NC16:0 ceramide. Thus, our method represents a unique procedure of simultaneous analysis of sphingomyelin and ceramide molecular species able to monitor the variation of the different pools in biological samples.     

Enzymatic deoxyglucosylation of ceramides by microsomes of BHK-21 cells. The effect of deoxyglucose treatment and herpesvirus infection

The microsomal fractions of cultured hamster fibroblasts (BHK-21 cells) catalyze the incorporation of glucose from UDPglucose or of deoxyglucose from UDPdeoxyglucose into a reaction mixture with liposomes consisting of ceramide and phosphatidylcholine. The microsomal fractions also catalyze the transfer of glucose from UDPglucose to endogenous acceptors. The specific activity of ceramide deoxyglucoside or ceramide glucoside formation was significantly higher when microsomal preparations obtained from deoxyglucose-treated or herpesvirus-infected BHK-21 cells were used as the glucosyltransferase source. Deoxyglucose was incorporated from UDPdeoxyglucose into hydroxy- and nonhydroxy-fatty acid-containing ceramides at approximately the same rate. Competitive inhibition of deoxyglucosylation of ceramides by UDPglucose suggests that both reactions were catalyzed by the same enzyme, viz. UDPglucose:ceramide glucosyltransferase. This inhibition of glycosphingolipid synthesis may account, in part, for the inhibitory effect of deoxyglucose on lipid-containing viruses.     

Occurrence of ungulic acid in some epidermal tissues

The lipids isolated from different animal tissues have been studied qualitatively, by TLC, for the occurrence of the ungulic acid fraction. This fraction was found in considerable amounts only in epidermal tissues and its keratinized derivatives. In the present study it was isolated from human keratinous epidermis, hair, and nails, pig bristles, wool, and feathers. The analytical results indicated that a lipid fraction from all of these sources contained ceramide, galactose, galactosamine, sulfate, and sialic acid in equimolar amounts, and that the fractions were similar to the ungulic acid isolated earlier from a horse's hoof.     

Effect of essential fatty acid deficiency on the epidermal sphingolipids of the rat

The epidermal sphingolipids from rats maintained on either a rat stock diet or a fat-free diet have been analyzed. Thin-layer chromatographic analyses have revealed glucosylceramides, acylglucosylceramides and four fractions of ceramides, one of which proved to be an acylceramide. The relative amounts of the glucosylceramides, acylglucosylceramides and acylceramides were increased in the essential fatty acid-deficient epidermis while one ceramide fraction was diminished. The other two ceramide fractions remained unchanged. The acylceramides and acylglucosylceramides from normal rat epidermis both contained long-chain omega-hydroxy acids in amide linkage to sphingosine bases and high proportions of linoleic acid in ester linkage. The linoleate, which is known to be crucial for the formation and maintenance of the epidermal water barrier, was replaced by oleate in the essential fatty acid-deficient rats.     

Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes

Transbilayer lipid motion in membranes may be important in certain physiological events, such as ceramide signaling. In this study, the transbilayer redistribution of lipids induced either by ceramide addition or by enzymatic ceramide generation at one side of the membrane has been monitored using pyrene-labeled phospholipid analogs. When added in organic solution to preformed liposomes, egg ceramide induced transbilayer lipid motion in a dose-dependent way. Short-chain (C6 and C2) ceramides were less active than egg ceramide, whereas dihydroceramides or dioleoylglycerol were virtually inactive in promoting flip-flop. The same results (either positive or negative) were obtained when ceramides, dihydroceramides, or diacylglycerols were generated in situ through the action of a sphingomyelinase or of a phospholipase C. The phenomenon was dependent on the bilayer lipid composition, being faster in the presence of lipids that promote inverted phase formation, e.g., phosphatidylethanolamine and cholesterol; and, conversely, slower in the presence of lysophosphatidylcholine, which inhibits inverted phase formation. Transbilayer motion was almost undetectable in bilayers composed of pure phosphatidylcholine or pure sphingomyelin. The use of pyrene-phosphatidylserine allowed detection of flip-flop movement induced by egg ceramide in human red blood cell membranes at a rate comparable to that observed in model membranes. The data suggest that when one membrane leaflet becomes enriched in ceramides, they diffuse toward the other leaflet. This is counterbalanced by lipid movement in the opposite direction, so that net mass transfer between monolayers is avoided. These observations may be relevant to the physiological mechanism of transmembrane signaling via ceramides.     

Dysregulated ceramide metabolism in mouse progressive dermatitis resulting from constitutive activation of Jak1

Coordinated lipid metabolism contributes to maintaining skin homeostasis by regulating skin barrier formation, immune reactions, thermogenesis, and perception. Several reports have documented the changes in lipid composition in dermatitis, including in atopic dermatitis (AD); however, the specific mechanism by which these lipid profiles are altered during AD pathogenesis remains unknown. Here, we performed untargeted and targeted lipidomic analyses of an AD-like dermatitis model resulting from constitutive activation of Janus kinase 1 (Spade mice) to capture the comprehensive lipidome profile during dermatitis onset and progression. We successfully annotated over 700 skin lipids, including glycerophospholipids, ceramides, neutral lipids, and fatty acids, many of which were found to be present at significantly changed levels after dermatitis onset, as determined by the pruritus and erythema. Among them, we found the levels of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine containing very long-chain (C22 or more) fatty acids were significantly downregulated before AD onset. Furthermore, in vitro enzyme assays using the skin of Spade mice demonstrated the enhancement of ceramide desaturation. Finally, we revealed topical application of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine before AD onset effectively ameliorated the progression of AD symptoms in Spade mice. Our results suggest that the disruption in epidermal ceramide composition is caused by boosting ceramide desaturation in the initiation phase of AD, which regulates AD pathogenesis.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.