biological membranes are mainly composed of phospholipids , sphingolipids , cholesterol , and membrane - associated proteins . these molecules are nonhomogeneously distributed in membranes and can rearrange , leading to the formation of membrane  domains  with highly differentiated molecular compositions and supramolecular architectures , which are stabilized by lateral interactions among the membrane components .
although glycosphingolipids ( gsls ) were originally thought to be structural components of plasma membranes , several experiments suggested that gsls are involved in the regulation of numerous cellular functions .
the membrane lipid bilayer is a stable structure , constituting a physical boundary between intra- and extracellular environments .
gsls are expressed on the surface of cellular membranes . based on their physicochemical properties , especially their many hydroxyl and acetamide groups , which can act as hydrogen bond donors and acceptors , gsls form clusters through cis interactions .
there is a general consensus on the roles played by the ceramide moiety of gsls in promoting the formation and stabilization of membrane lipid domains .
in addition , ceramide was also shown to be involved in gsl - mediated functions and several biological activities [ 3 , 4 ] .
we recently showed that very long fatty acid chains of ceramide , such as c24:0 and c24:1 , are responsible for the direct connection between lactosylceramide ( laccer , cdw17 ) and palmitoylated signal transducer molecules .
moreover , the phosphorylated product of sphingosine , sphingosine-1-phosphate ( s1p ) , was shown to be important in immunological , especially inflammatory reactions [ 4 , 6 ] .
this review describes the role of the fatty acid chains of ceramide in gsl - mediated outside - in signaling in promoting gsl - enriched domain - mediated cellular functions , as well as the activities of s1p in inflammatory reactions of keratinocytes in human .
the most studied gsl - enriched domains are membrane lipid microdomains , called lipid rafts , defined by their gsl- and cholesterol - rich nature , enrichment in gpi - anchored proteins and membrane - anchored signaling molecules , and cytoskeletal association [ 7 , 8 ] .
as shown in artificial membrane models , gsls tend to form clusters , with this cluster formation confirmed in intact cells by immunoelectron microscopy [ 1012 ] .
the gsl - enriched microdomains on plasma membranes have a diameter of 50100  nm and include signal transducer molecules , such as src family kinases [ 11 , 12 ] .
gsls that contain saturated fatty acid chains with higher transition temperatures   show ordered , less fluid , liquid phase .
cholesterol is composed of a highly hydrophobic sterol - ring system and 3-hydroxy moiety , the only hydrophilic part of the molecule . the small cholesterol sterol - ring system and the ceramide moiety of sphingolipids are thought to interact via hydrogen bonds and hydrophobic van der waal 's interactions .
in addition , hydrophilic interactions between sugar moieties of gsls promote the lateral association of gsls and cholesterol .
phospholipids tend to be loosely packaged in bilayers , resulting in the formation of liquid - disordered membranes that allow rapid lateral and rotational movement of lipids .
these interactions result in the separation of gsl- and cholesterol - enriched lipid microdomains from other phospholipids in the cell membrane and the formation of distinct domains .
electron microscopy using labeled anti - gsl antibodies has revealed gsl clusters on the surface of glycosphingolipid / phosphatidylcholine ( pc ) liposomes , even in the absence of sphingomyelin ( sm ) and cholesterol .
the anti - laccer mabs t5a7 and huly - m13 recognized laccer on human neutrophils , but only t5a7 recognized laccer on mouse neutrophils .
interestingly , huly - m13 but not t5a7 can be used for immunoprecipitation , suggesting a difference in binding and/or cluster formation of huly - m13 and t5a7 to laccer - enriched microdomains .
indeed , stimulated emission depletion ( sted ) superresolution microscopy showed that t5a7 and huly - m13 bind to different regions of the same laccer / dioleoylphosphatidylcholine ( dopc ) liposomes ( figure 1 ) .
laccer - enriched microdomains are composed of laccer , sm , phospholipids , and cholesterol .
surface plasmon resonance analysis showed that reduction of the laccer content in the dopc / cholesterol / laccer / sm lipid layer markedly decreased huly - m13 but not t5a7 binding to laccer , suggesting that the content of laccer in laccer - enriched microdomains affects the binding avidity of huly - m13 to laccer .
in contrast , the molecular species of pc , including dopc , dipalmitoylphosphatidylcholine ( dppc ) , and palmitoyl - oleoyl - phosphatidylcholine ( popc ) , did not affect the binding avidity of huly - m13 to laccer - coated plastic wells .
lactose inhibited the binding of huly - m13 to laccer / dopc liposome - coated and dopc / laccer mixture - coated plastic wells , suggesting that huly - m13 binds only to laccer clusters in laccer - enriched microdomains .
in contrast , the binding avidity of t5a7 to laccer - coated plastic wells was much weaker than its binding avidity to dopc / laccer- , popc / laccer- , and dppc / laccer mixture - coated wells , suggesting that the binding of t5a7 to laccer is affected by pc . the ability of lactose to inhibit the binding of t5a7 to dopc / laccer liposome - coated plastic wells was similar to its ability to inhibit the binding of huly - m13 .
in contrast , lactose inhibition of t5a7 binding to dopc / laccer mixture - coated plastic wells was significantly lower than its inhibition of huly - m13 binding , suggesting that t5a7 recognizes the pc - enhanced three - dimensional structure of laccer clusters .
thus , huly - m13 may bind to the core region of lactose clusters in laccer - enriched domains , while t5a7 binds to  dispersed  laccer clusters in the phase boundary regions of these microdomains .
these findings suggest that the specificities of these antibodies against the same gsls are dependent on the organizations of the gsls and molecules surrounding the gsl - enriched domains .
disorders of the degradation of gsls sometimes cause human diseases [ 18 , 19 ] . for degradation ,
, those molecules are constitutively degraded by their suitable catabolic enzymes . when the activities of lysosomal enzymes are impaired , degradation is not able to proceed normally and undegraded molecules accumulate in the organelle and intracellular membranes , causing several metabolism diseases .
for instance , genetic disorder of glucocerebrosidase ( gba ) ( ec 3.2.1.45 ; ) , gaucher disease , results in accumulation of glccer and its deacetylated form glucosylsphingosine is caused by abnormality of gba .
gaucher disease is a multisystem disorder whose features include peripheral blood cytopenias , hepatosplenomegaly , bone disease , and neurological manifestations in some cases .
the form of intravenous enzyme replacement therapy in the 1990s has been developed and resulted in dramatic improvements in haematological and visceral disease .
recognition of complications , including multiple myeloma and parkinson disease , has challenged the traditional macrophage - centric view of the pathophysiology of this disorder .
however , the pathways by which enzyme deficiency results in the clinical manifestations of this disorder remain obscure . in spinal cords of amyotrophic lateral sclerosis ( als ) patients , levels of gm1 , gm3 , laccer , glccer , galcer , and ceramide were significantly elevated .
furthermore , glucocerebrosidase-1 , glucocerebrosidase-2 , hexosaminidase , galactosylceramidase , -galactosidase , and -galactosidase activities were also elevated in those patients .
inhibition of glucosylceramide synthesis accelerated disease course in als model mice , whereas infusion of exogenous gm3 significantly slowed the onset of paralysis and increased survival .
these observations suggest that gsls and their metabolism are likely important participants in pathogenesis of als .
further studies about gsl metabolism pathways in gsl - related disease will serve to advance our understanding of other associated disorders .
over the last 30 years , many studies have indicated that gsls expressed on the cell surface may act as binding sites for microorganisms .
the binding avidities of microorganisms to several types of gsl [ 2427 ] suggest that gsls are involved in host - pathogen interactions .
indeed , microorganisms have been shown to recognize and enter host cells via gsl - enriched membrane microdomains on the cells . among gsls
, laccer has been well described to bind to several kinds of microorganisms , including viruses and fungi .
for instance , candida albicans specifically bind to laccer though the binding of -1,6-long glucosyl side - chain - branched -1,3-glucan to laccer - enriched domains [ 26 , 29 ] .
it is also well known that microorganisms - derived toxins , such as shiga toxin , specifically bind to gsls [ 3032 ] .
furthermore , a sphingolipid metabolite , ceramide , has been demonstrated to play a crucial role in pulmonary infection and inflammation .ceramide , which is degraded product of gsls and sphingomyelin , has been reported to form ceramide - rich membrane platforms and involve uptake of several microorganisms including pseudomonas aeruginosa .
abnormal amounts of enzymes involved in the synthesis of ceramide have been demonstrated in emphysematic smokers and in patients with severe sepsis .
gsls have been reported to interact with membrane proteins and modulate the properties of these proteins [ 2 , 25 ] .
in addition , certain proteins , including glycosylphosphatidylinositol- ( gpi- ) anchored and palmitoylated proteins , tend to enter gsl - enriched membrane microdomains .
however , the mechanism by which gsls interact with proteins and mediate outside - in signaling is unclear .
the ceramide moiety consists of a long chain base linked to a fatty acid chain .
sphingosine containing c18 carbons [ ( 2s,3r,4e)-2-amino-1,3-dihydroxy - octadecene ] is generally the main structure in mammals , but a structure containing 20 carbons is relatively abundant in neurons .
ceramide is synthesized by ceramide synthases ( cers ) 16 , each of which uses a restricted subset of fatty acyl - coas for n - acylation of the sphingoid long chain base .
the expression levels of genes encoding cers are tissue specific , suggesting that the molecular varieties and expression patterns of gsls are associated with the functions of these cells .
although gsl - enriched microdomains have been implicated in a number of important membrane events [ 2 , 38 , 39 ] , the molecular mechanisms responsible for gsl - mediated cell functions are still unclear .
one of the main issues centers around the association of gsls with signal transducer molecules localized on the cytosolic side .
laccer , along with the src family kinase lyn , forms lipid microdomains on the plasma membranes of human neutrophils and is involved in several cellular functions , including chemotaxis , phagocytosis , and superoxide generation , highly dependent on lyn [ 16 , 29 , 38 ] .
hl-60 cells differentiated into neutrophilic lineage cells by dmso ( d - hl-60 ) were found to acquire superoxide generating activity , but not through laccer , despite their expression of laccer on plasma membranes .
most laccer and lyn were recovered in the microdomain fractions of neutrophils and d - hl-60 cells .
lipidomics analysis revealed that laccer in the neutrophil plasma membrane was mainly composed of molecular species containing c16:0 , c24:1 , and c24:0 fatty acid chains , whereas over 70% of laccer in the plasma membranes of d - hl-60 cells contained c16:0 fatty acid chains , but only about 14% were c24:1 and c24:0 .
lyn was immunoprecipitated by anti - laccer antibody in neutrophils but not d - hl-60 cells .
importantly , lyn was coimmunoprecipitated by anti - laccer antibody from the detergent resistant membrane ( drm ) fraction of plasma membranes from c24:0 and c24:1 , but not c16:0 or c22:0 , laccer - loaded d - hl-60 cells .
anti - laccer antibody induced superoxide generation from d - hl-60 cells loaded with c24:0-laccer , but not c16:0-laccer .
lyn colocalized with laccer - enriched domains of d - hl-60 cells loaded with c24:0-laccer , but not c16:0-laccer .
these results suggested that the c24 fatty acid chain of laccer is indispensable for connecting lyn with laccer - enriched microdomains .
knockdown of lyn molecules by human lyn - specific short interfering rna ( sirna ) in d - hl-60 cells completely abolished the effects of c24:1-laccer loading function , suggesting that lyn is crucial for c24-laccer - mediated neutrophil function .
experiments using azide - photoactivatable tritium - labeled c24- and c16-laccer revealed that c24- but not c16-laccer directly associated with lyn and a heterotrimeric g protein subunit gi .
these results confirm a specific direct interaction between c24-laccer and the signal transduction molecules lyn and gi , which are associated with the cytoplasmic layer via palmitic acid chains ( figure 2 ) .
laccer species with very long fatty acids are indispensable for lyn - coupled laccer - enriched membrane microdomain - mediated neutrophil functions .
gpi - anchored proteins are composed of glycerol phospholipids , which do not have c24 fatty acid chains , suggesting that gpi - anchored proteins are not able to form large clusters by themselves and can not directly connect with signal transduction molecules through fatty acid chains . to mediate cell functions , gpi - anchored proteins require signal transduction molecule - coupled transmembrane proteins or gsl - enriched domains , such as laccer - enriched domains .
the epidermis consists of a single layer of proliferating undifferentiated keratinocytes , the stratum basale , and several superficial layers of the stratum spinosum and stratum granulosum ( sg ) , which form the stratum corneum ( sc ) .
the sc acts as an air - liquid interface barrier to avoid drying of tissues in contact with air .
ceramide is the main component of sc and is important for the water retention and permeability barrier functions of sc .
sphingosine can be phosphorylated by sphingosine kinase to form s1p , a molecule involved in a wide range of cellular functions , including growth , differentiation , survival , chemotaxis , angiogenesis , and embryogenesis , in various types of cells [ 44 , 45 ] .
s1p was shown to inhibit keratinocyte proliferation , to promote corneocyte differentiation , and to chemoattract keratinocytes .
roles of s1p in skin immunological functions have been demonstrated in mouse models [ 45 , 4751 ] .
mice are the good experimental tool of choice for the majority of immunologists , and the study of immune responses in mice has provided considerable insight into human immune system function . however , there are significant differences in immunological reactions between mice and human
. little is known , however , about the role of ceramide metabolites in the immunological functions of differentiating keratinocytes .
a neutral cdase from pseudomonas aeruginosa an17 ( pacdase ) isolated from a patient with atopic dermatitis ( ad ) was shown to require detergents to hydrolyze ceramide .
staphylococcus aureus - derived lipids , which consist primarily of cardiolipin and phosphatidylglycerol , enhanced the pacdase hydrolysis of normal ceramide and of human skin - specific omega - hydroxyacyl ceramide in the absence of detergents .
a three - dimensionally cultured human primary keratinocyte ( 3d keratinocyte ) culture system has been utilized to simulate epidermal differentiation at its air - liquid interface , resulting in the generation of basal , spinous , and granular layers and an sc , with the latter displaying permeability barrier functions .
treatment of 3d keratinocytes with pacdase and water - soluble stimulants of keratinocytes , including trypsin , dermatophagoides pteronyssinus class 1 allergen ( der p1 ) , and dermatophagoides farinae allergen ( der f1 ) had no effect on the expression of any of the genes in our dna microarray analysis , indicating that the sc of the 3d keratinocyte culture acts as a permeability barrier .
triton x-100 is a detergent that reduces permeability barrier functions , thereby moderately increasing transepidermal water loss and the production of erythema on human skin . in the presence of 0.1% triton x-100 , pacdase
markedly enhanced tnf- mrna expression in 3d keratinocytes , an increase not observed in cells treated with triton x-100 alone .
tnf- mrna expression was not enhanced by heat - inactivated or mutant pacdase , suggesting that ceramide metabolites induce tnf- mrna expression in keratinocytes .
tnf- , a critical cytokine in several dermatological diseases , is secreted by keratinocytes   and shown to be involved in the progression of atopic dermatitis ( ad ) . among the metabolites of ceramide ,
s1p is synthesized from sphingosine by sphingosine kinase ( sphk ) and stimulates 3d keratinocytes through specific receptors .
both the specific sphk inhibitor cas 1177741 - 83 - 1 and the s1p receptor antagonist vpc 23019 suppressed the pacdase - induced expression of tnf- mrna in 3d keratinocytes .
s1p is generally considered to stimulate cells through plasma membrane g protein - coupled receptors , for example , s1p1s1p5 .
however , vpc2301 , a competitive antagonist for s1p1 and s1p3 receptors , inhibited the pacdase - enhanced gene expression not only of tnf- but also of endothelin-1 and il-8 .
thus , the s1p - induced production of these inflammatory mediators is mediated by s1p receptors in human primary keratinocytes in a 3d culture system .
cdna microarray analysis showed that s1p strongly upregulated the expression of endothelin-1 , cxcl1 , tnf- ,
-defensin 5 , il-8 , cxcl2 , interferon regulatory factor 1 , gadd45 gamma , and il-23 subunit mrnas .
il-8 , cxcl1 , and cxcl2 have been reported to be upregulated in the lesional skin of patients with ad and psoriasis .
s1p also enhanced the expression of claudin-4 mrna , which has been observed in more layers of psoriatic than normal epidermis .
tnf- can induce the production of endothelin-1 and il-8 by human keratinocytes [ 66 , 67 ] .
infliximab , a chimeric igg1 monoclonal antibody against human tnf- , inhibits the tnf--mediated production of il-8 by keratinocytes .
the nf-b inhibitor curcumin inhibited pacdase - induced expression of il-8 and endothelin-1 mrnas but not of tnf- mrna .
therefore , it is likely that s1p induces tnf- production and release from 3d keratinocytes via s1p receptors , resulting in tnf- induction of cytokine production through nf-b - mediated signal transduction ( figure 3 ) .
tnf- is a critical cytokine in psoriatic immunopathology , and the development of an effective strategy is required to counteract its effects .
infliximab , which is used to treat patients with plaque psoriasis , psoriatic arthritis , pustular psoriasis ( excluding localized type ) , and psoriatic erythroderma [ 71 , 72 ] , downregulates antiapoptotic proteins in regressing psoriatic skin .
the effects of infliximab have also been evaluated in other inflammatory dermatoses and in systemic diseases involving the skin , pityriasis rubra pilaris , pyoderma gangrenosum , and cutaneous sarcoidosis .
ad is characterized by a marked reduction in ceramides in the sc of lesional and nonlesional forearms [ 74 , 75 ] and by increased activities of the enzymes ceramidase ( cdase ) .
the metabolic conversion of ceramide to s1p has been found to protect keratinocytes against uvb - induced , ceramide - mediated apoptosis .
histamine h1-receptor blockers are used to treat all types of itch resulting from serious skin diseases , such as ad , as well as from renal and liver diseases .
however , they often lack efficacy in chronic itch , a profound clinical problem that decreases quality of life . nerve density in the epidermis is partly involved in itch sensitization in pruritic skin diseases , such as ad . endothelin-1 has been shown to elicit itch in humans [ 8082 ] .
the molecular pathways that contribute to the transduction of itch responses to endothelin-1 do not require either plc3 or trpv1 of neurons , which mediate histamine- and serotonin - induced itch responses , respectively .
several reports have described the roles of ceramide metabolites in immunological and inflammatory diseases [ 8488 ] . however , the physiological roles of gsl - enriched microdomains are largely undetermined , although much is known about the organization and functions of laccer - enriched microdomains [ 24 , 89 , 90 ]
. the analogous patterns of gsls and motifs of pamps result in the generation of autoantibodies against these gsls , inducing severe autoimmune inflammatory diseases .
antibodies against neuronal tissues are involved in immune - mediated neurological disorders , with expression of several of these antibodies found to correlate with the pathophysiology of these diseases [ 92 , 93 ] .
therefore , elucidation of their organization and structural specificities , based on interactions between gsls and surrounding molecules , are important for understanding the physiological functions of gls - enriched microdomains and their related diseases .
 glycosphingolipids ( gsls ) are composed of hydrophobic ceramide and hydrophilic sugar chains . 
 gsls cluster to form membrane microdomains ( lipid rafts ) on plasma membranes , along with several kinds of transducer molecules , including src family kinases and small g proteins . 
 however , gsl - mediated biological functions remain unclear . 
 lactosylceramide ( laccer , cdw17 ) is highly expressed on the plasma membranes of human phagocytes and mediates several immunological and inflammatory reactions , including phagocytosis , chemotaxis , and superoxide generation . 
 laccer forms membrane microdomains with the src family tyrosine kinase lyn and the gi subunit of heterotrimeric g proteins . 
 the very long fatty acids c24:0 and c24:1 are the main ceramide components of laccer in neutrophil plasma membranes and are directly connected with the fatty acids of lyn and gi . 
 these observations suggest that the very long fatty acid chains of ceramide are critical for gsl - mediated outside - in signaling . 
 sphingosine is another component of ceramide , with the hydrolysis of ceramide by ceramidase producing sphingosine and fatty acids . 
 sphingosine is phosphorylated by sphingosine kinase to sphingosine-1-phosphate , which is involved in a wide range of cellular functions , including growth , differentiation , survival , chemotaxis , angiogenesis , and embryogenesis , in various types of cells . 
 this review describes the role of ceramide moiety of gsls and its metabolites in immunological and inflammatory reactions in human . 
1. Introduction
2. Organization of GSL-Enriched Lipid Microdomains
3. GSL Metabolism Diseases
4. GSL- and Ceramide-Enriched Membrane Microdomains Are Binding Targets for Pathogenic Microorganisms
5. Fatty Acid Chains of Ceramide Are Indispensable for GSL-Mediated Signaling
6. Role of Ceramide Metabolite Sphingosine-1-phosphate in Immunological Reactions of Human Keratinocytes
7. Conclusion