Epidermal Lamellar Granules

A typical lamellar granule (LG) with a diameter of about 200 nm is shown in Figure 1 (review [1]). The internal contents of the LG appear to be pairs of bilayers separated by a central minor electron dense band and bounded by major electron dense bands. The distance from the center of a major electron dense band to the center of the next electron dense band is 9.6 nm [2]. LGs were first described by Selby [3], who thought they were degenerating mitochondria. Subsequently, they were described in detail by Odland [4] and others [5-12]. They were recognized as unique organelles found in keratinizing epithelia and nowhere else. Collectively, these early studies [5-12] established that LGs are membrane-bound and contain one or several tightly packed stacks of bilayer membranes, thought to be derived from flattened and stacked lipid vesicles [1]. LGs first appear in the spinous layer, accumulate in the granular layer, and extrude their internal contents into the intercellular space at the interface between the stratum granulosum and stratum corneum. With slight variation in morphology, LGs are found in the epidermis of mammalian, avian, and reptilian species. Based on transmission electron microscopy (TEM) images, it has been proposed that LGs bud off from the Golgi apparatus [6].

Production of LGs, revealed by TEM, has been used as an indicator of differentiation in keratinocyte cultures [13-21]. Formation of normal LGs has been considered a hallmark of differentiation in organ culture [13], stratified keratinocyte cultures [16, 17, 19-21], and organotypic skin cultures [14, 15, 18].

In order to determine lipid composition and associated enzymes, several groups have isolated LG-enriched fractions based on their low buoyant density [22-26] or their fairly unique size [ 24, 27]. In the fractionation based on size, an epidermal homogenate is passed through a series of nucleopore filters of decreasing pore sizes (8.0, 3.0, 1.0, 0.8, 0.6, and 0.4 µm). Since LGs are about 200 nm in diameter, they will pass through this series of filters while all larger material will be removed. It should be pointed out that LG fractions prepared on the basis of either buoyant density or size are not pure but are enriched in LGs [27].

Direct lipid analyses [23, 24] of isolated LGs from rodent and pig epidermis have demonstrated that these organelles contain phospholipids, cholesterol, and glycolipids as major components, and small proportions of fatty acid, cholesterol esters, and ceramides.

The glycolipids are mainly glucosylceramides, the most unusual of which is a linoleate-rich acylglucosylceramide [28-31], which has been implicated in the as-sembly of LGs [32]. This acylglucosylceramide consists of 30- to 34-carbon ω-hydroxyacids amide-linked to sphingosine and dihydrosphinmgosine bases, with glucose β-glycosidically attached to the primary hydroxyl group of the long-chain base and linoleic acid ester-linked to the ω-hydroxyl group (Fig. 2, center).

The 2 strikingly unusual features of this molecule are the very long ω-hydroxyacyl chains, which are long enough to completely span a typical bilayer, and the enrichment in linoleic acid. Linoleic acid is an essential fatty acid without which barrier function deteriorates, and the acylglucosylceramide is the major carrier of linoleic acid in the viable portion of the epithelium. It has been proposed that the ω-hydroxyacyl portion of this molecule does span a bilayer while the linoleate tail inserts into an adjacent region of bilayer, thus riveting the 2 regions of bilayer together at a molecular level [32]. This sort of interaction could promote the flattening and stacking of lipid vesicles in the formation of the LG contents. This “molecular rivet hypothesis” is supported by experiments in which acylglucosylceramide has been shown to promote the aggregation and flattening of synthetic lipid vesicles [33]. It is also supported by the coincidental occurrence of acylglucosylceramide and LGs in all of the keratinizing epithelia and the absence of both acylglucosylceramide and LGs in other tissues, and by the coincidental occurrence of acylglucosylceramide and LGs in the epidermis of terrestrial mammals, birds, and reptiles combined with their absence from the epidermis of amphibians and fish [34].

Isolated epidermal LGs have been shown to contain a number of hydrolytic enzymes including β-glucosidase, acid phosphatase, phospholipase A, sphingomyelinase, carboxypeptidase, and cathepsin B [24, 35]. They have also been shown to contain ceramide glucosyltransferase, which is considered a biochemical marker for the Golgi [25], and caveolins, which are scaffolding proteins involved in the formation of cholesterol- and sphingolipid-rich membrane domains [36]. Sterol sulfatase is not enriched in LGs.

Raymond et al. [26] isolated a human epidermal LG-enriched fraction and identified 984 proteins by high-throughput mass spectrometry. Thirty-one of these corresponded to lysosomal components, including lipid-processing enzymes noted previously. Other identified components include proteolytic enzymes and protease inhibitors, which together could regulate desquamation. One identified protein, CLIP-170, is known to mediate interaction between organelles and microtubules. This may be involved in the movement of the LGs to the apical end of the cell prior to exocytosis of the contents. This study verified the previous findings of Freinkel and Traczyk [22, 35] demonstrating the presence of glucocerebrosidase, sphingomyelinase, and prospholipase A in the LG-enriched fractions. These hydrolytic enzymes are thought to convert glucosylceramides and sphingomyelin into ceramides and to liperate fatty acids from the phosphoglycerides.

Antimicrobial peptides produced in the epidermis are a critical part of innate immunity of the skin [37, 38]. Defensins and cathelicidins are among the broad-acting antimicrobial peptides produced in the epidermis. They are small (12–50 amino acids), positively charged, and have a hydrophobic domain [39, 40]. The positive charge allows these molecules to bind to the negatively charged phospholipids. The hydrophobic tail then inserts into the bacterial membrane, causing leakage and cell death. Defensins and cathelicidin were among the peptides identified in the study by Raymond et al. [26]; however, it was possible that these peptides were present as contaminants in the LG preparation. It has been convincingly demonstrated that keratinocytes store cathelicidin in LGs [41]. More research is needed in this area.

Although there is considerable evidence that acylglucosylceramide plays a role in assembling the internal contents of LGs, it has been estimated that about two-thirds of the LG-associated acylglucosylceramide in the epidermis is in the bounding membrane of the organelle [42]. This is based, in part, on the relative amounts of covalently bound ω-hydroxyceramide and acylceramide found in the stratum corneum. The pool of acylglucosylceramide in the bounding membrane of the LG is introduced to the cell surface when the bounding membrane of the LG fuses into the cell plasma membrane and serves as the precursor of the covalently bound hydroxyceramide (Fig. 2, top). Acylglucosylceramide associated with the internal lamellae of the LG will be extruded and converted into acylceramide (Fig. 2, bottom), which will pass into the intercellular spaces of the stratum corneum.

Given the known sidedness of ceramide glucosyltransferase in the Golgi, it is expected that the glucosyl moiety of acylglucosylceramide in the LG would be on the cytosolic surface, and it is typical for glycolipids in closed vesicular membranes to be oriented with the glucose on the outside. However, this orientation of acylglucosylceramide would position it with the wrong end of the molecule facing the nascent cornified envelope for subsequent attachment of the ω-hydroxyceramide to the envelope proteins, which occurs exclusively via the ω-hydroxyl groups [43]. The action of a flipase at about the time of LG fusion and extrusion could lead to ω-hydroxyceramide with the appropriate orientation. It is speculated that acylglucosylceramide does arrive in the plasma membrane with the glucosyl moiety on the cytoplasmic side, and that the ester-linked fatty acid can be removed through the action of an acid lipase following 2 specific lipoxygenase attacks [44] at the acidic interface between the granular and cornified layers. Flipase action at this time could both expose the glucosyl moiety to β-glucocerebrosidase at the interface and bring the hydroxyl group into apposition with the nascent cornified envelope proteins to which it becomes attached.

Were acylglucosylceramide molecules in the Golgi to be translocated to orient the glucosyl moiety to the luminal side, additional glycosylation would be expected because the enzymatic mechanism for this is at this location. However, a mechanism that avoids additional glycosylation in the epidermis cannot be ruled out. Flipase action at the time of LG formation would place the glucose moieties on the inside of the LG bounding membrane. Although unusual, this orientation could possibly explain several observations. For one thing, this orientation would place the appropriate ω-hydroxyl end of acylglucosylceramide adjacent to the cornified envelope for lipid envelope formation. The glucose moieties on the inside of the LG could hydrogen bond with water, effectively reducing the activity of free water inside the LG. This could explain the observation that the LGs are uniquely stable to freezing and thawing [25]. It could also be one factor in explaining the fact that the various hydrolases do not extensively act on their lipid substrates until they are extruded into the extracellular space. Another possibility raised by this orientation of acylglucosylceramide in the LG bounding membrane is that the linoleate tails of the acylglucosylceramide molecules would be in the outer portion of the bounding membrane. As the bounding membrane of the LG comes into close apposition with the inner surface of the cell plasma membrane, the linoleate could extend and insert into the inner leaflet of the plasma membrane. This could locally destabilize the membrane system and facilitate fusion. A similar mechanism has been proposed for the fusion of more conventional phospholipids membranes [45]. Flipases are known in other systems [46, 47] but have not been reported in the epidermis. The above discussion of a possible role for a flipase is speculative; however, one of the most studied flipases is an ATP-binding cassette (ABC) transporter (ABCA4) [47], reminiscent of the LG-associated ABCA12 [48].

There is considerable evidence that LGs arise from the Golgi apparatus. As noted above, this suggestion was first based on TEM images [7]. A number of biochemical markers of the Golgi have been shown to be associated with LGs including ceramide glucosyltransferase [25], ABCA12 [48, 49], and Rab11 [50].

The Golgi pH regulator (GPHR) is an anion channel that serves to acidify the lumen of the trans-Golgi and is essential for Golgi function [48, 49, 51]. In the GPHR knockout mouse, LG formation is markedly impaired and barrier function is diminished [52]. In addition to structurally aberrant LGs and increased TEWL, the epidermis of these mice contained less cathelicidin-related antimicrobial peptide (CRAMP).

In one study, the uppermost granular cells were specifically examined [53]. After staining with Nile red or NDB-ceramide, confocal microscopy revealed a tubule-reticular membrane system in the apical cytosol of neonatal and adult mouse epidermis. This same trans- Golgi network-like structure was found in the uppermost granular cells in organotypic keratinocyte cultures. When the cultures were challenged by changing the calcium concentration of the medium, it appeared that LGs budded off from the trans- Golgi network-like structure.

A number of LG defects have been shown to result in skin diseases characterized by diminished barrier function, generally measured as increased TEWL. These diseases are generally characterized by some degree of ichthyosis, the severity of which ranges from mild to life-threatening.

ABCA12 is an ABC transporter that uses ATP for energy necessary to transport lipids across membranes or into vesicles and is associated with epidermal LGs [49]. Mutations of the gene coding for this transporter are responsible for harlequin ichthyosis [53]. In this condition, the LGs are absent or grossly abnormal [54]. This results in severe reduction of lipids in the intercellular spaces of the stratum corneum and diminished barrier function [49, 55]. Cornified envelope proteins and keratins are not affected [56]. Other mutations of this same gene may result in lamellar ichthyosis or congenital ichthyosiform erythroderma [55, 57].

Mutations of the gene VPS33B results in arthrogryptosos, renal disfunction and cholestasis syndrome (ARC). Patients with ARC suffer severe ichthyosis. In this condition, LGs have abnormal morphology and exocytosis is defective [58, 59].

Hyperkeratosis in atopic dermatitis lesions has been attributed to incomplete secretion of the serine protease KLK 7 from LGs and upregulation of the serine protease inhibitor LEKTI [60].

A mutation of CGI-58 protein has been shown to cause Dorfman-Chanarin syndrome, one characteristic of which is ichthyosis. The CGI-58 protein is involved in triglyceride breakdown and, with this, the mutant protein lipid droplets accumulate in the cytosol of the keratinocyte. Electron microscopy has revealed that the LG contents are abnormal, and it has been suggested that this defect may lead to the ichthyotic condition [61]. Likewise, a specific lipoxygenase mutation has been shown to cause incomplete LG secretion, leading to congenital ichthyosaform erythroderma [62]. Both Dorfman-Chanarin syndrome and congenital ichthyosaform erythroderma are autosomal recessive diseases.

Anything that significantly alters LG contents or interferes with their exocytosis is likely to result in diminished permeability barrier function, attributed to decreased or inappropriate lipids in the intercellular spaces of the stratum corneum, defective desquamation due to failed or diminished delivery of proteases required for desquamation, or increased susceptibility to infection due to diminished delivery of antimicrobial peptides. In a number of skin diseases with impaired barrier function, the barrier has been at least partially restored by topical application of ceramides or combinations of ceramides plus cholesterol and fatty acids [63-71].

In 2003, Norlen et al. [72] published a cryo-TEM study of skin barrier formation in which they described a tubuloreticular network, similar to that described by Elias et al. [53], in the secretory granular cell after using a microwave-enhanced fixation technique. There were no discrete LGs. This was further elaborated upon by den Hollander et al. [73].

Ishida-Yamamoto et al. [74] imaged a number of LG markers by confocal laser scanning microscopy and by immunoelectron microscopy. These markers included glucosylceramides, cathepsin D, corneodesmosin, kallikrein, and KLK8. They concluded that their results were consistent with a tubuloreticular system rather than discrete LGs.

The findings of Elias et al. [53] suggest that, in an unstimulated state, the tubuloreticular network predominates and individual LGs may be sparse, at best. However, under stress to stimulate lipid secretion, individual LGs may bud off from this network. Some kinds of chemical fixation may acutely provide such a stress.

Traditional electron microscopic studies, including TEM and the freeze-fracture method, have revealed LGs as small organelles in keratinizing epithelia. They are round to ovoid in shape with a diameter of about 200 nm and consist of a unit-bounding membrane surrounding stacks of internal membranous disks. They first appear in the spinous layer, increase throughout the granular layer, and extrude their contents at the boundary between the granular layer and the stratum corneum. Studies of isolated LGs support their origin from the Golgi. Isolated LGs contain sphingomyelin and glucosylceramides, and also the hydrolytic enzymes necessary to convert these lipids into ceramides. LGs also deliver proteolytic enzymes necessary for desquamation and antimicrobial peptides to the intercellular space. Mutations of ABCA12 result in abnormal LGs and lead to either harlequin ichthyosis, lamellar ichthyosis, or congenital ichthyosiform erythroderma. Several other mutations that alter LGs also result in ichthyotic conditions. Recent work with cryo-TEM suggests that LGs may not exist as discreet organelles. Instead, it is proposed that there is membrane continuity from the Golgi through the intercellular spaces of the stratum corneum.

Additional work is needed to determine whether the view of LGs based on traditional TEM and freeze-fracture electron microscopy is valid or needs to be replaced with the continuous membrane model based on cryo-TEM.

The author would like to acknowledge Nancy Wertz for editorial assistance in the preparation of the manuscript.

The author has no conflict of interest with respect to this work.