Altered Lamellar Body Secretion and Stratum Corneum Membrane Structure in Netherton Syndrome: Differentiation From Other Infantile Erythrodermas and Pathogenic Implications

Background  The infant with Netherton syndrome (NS) typically displays a generalized erythroderma covered by fine, translucent scales, which can be difficult to distinguish clinically from erythrodermic psoriasis, nonbullous congenital ichthyosiform erythroderma, or other infantile erythrodermas. Some infants with NS develop progressive hypernatremic dehydration, failure to thrive, and enteropathy. Such complications can be fatal. Diagnosis is typically delayed until the appearance of a pathognomonic hair shaft anomaly, trichorrhexis invaginata (bamboo hair). To facilitate the early diagnosis of NS, we obtained biopsy specimens from 7 patients with erythrodermic NS and compared their morphologic findings to those of 3 patients with erythrodermic psoriasis and 2 with congenital ichthyosiform erythroderma. Biopsy specimens were processed for light and electron microscopy using postfixation with osmium tetroxide and ruthenium tetroxide.

Observation  In NS, and often in congenital ichthyosiform erythroderma and erythrodermic psoriasis, the stratum corneum layer was largely replaced by parakeratotic cells. A distinctive feature—premature secretion of lamellar body contents—occurred only in NS. Furthermore, lamellar body–derived extracellular lamellae and stratum corneum lipid membranes were separated extensively by foci of electron-dense material. Finally, transformation of lamellar body–derived lamellae into mature lamellar membrane structures was disturbed in NS.

Conclusions  Premature lamellar body secretion and foci of electron-dense material in the intercellular spaces of stratum corneum, features not observed in other erythrodermic disorders, appear to be frequent and relatively specific markers for NS. These ultrastructural features could permit the early diagnosis of NS before the appearance of the hair shaft abnormality. These abnormalities could explain the impaired permeability barrier in NS, and account for hypernatremia and dehydration in infants with NS.

NETHERTON syndrome (NS) is a rare autosomal recessive disorder of cornification, characterized by the triad of ichthyosis, hair shaft defects, and atopy. The nature of the ichthyosiform dermatosis in this syndrome has been the subject of considerable debate, raising the question of whether NS is a heterogeneous condition.¹ Although some patients with NS reveal a distinctive ichthyosiform phenotype of erythematous, polycyclic plaques with "double-edged" scale (ichthyosis linearis circumflexa [ILC]), this feature usually appears after infancy,²as do pili torti, trichorrhexis nodosa, and the pathognomonic trichorrhexis invaginata. Most infants with NS display a generalized exfoliative erythroderma, with or without an atopic diathesis.^3,4Recent studies suggest that ILC and erythroderma/dermatitis represent different phases of the same disease.^5,6 Although ILC is the predominant cutaneous manifestation in older patients with NS,^5,7-10erythrodermic infants with NS are often misdiagnosed as having the following: (1) another metabolic disease with generalized dermatitis, such as immunodeficiency disorder with dermatitis (so-called Leiner disease) or acrodermatitis enteropathica; (2) a severe form of more common disorders, such as erythrodermic psoriasis (PsoE)¹¹; or (3) another recessive disorder of cornification, eg, nonbullous congenital ichthyosiform erythroderma (CIE).^1,4,12,13 Because ILC and its characteristic hair shaft abnormalities typically do not become evident until after the first year of life, a definitive diagnosis of NS is usually delayed.⁶

A high rate of morbidity and mortality accompanies the erythroderma in infants with NS.^5,6 Although some authors^5,14 suggest that systemic complications of NS could be due to a severe, underlying immunodeficiency, recent studies⁶ demonstrate that, apart from markers of atopy such as increased IgE, significant immune defects are not present. Others^5,15-17have proposed that increased rates of transcutaneous water loss, resulting in hypernatremic dehydration and hypothermia in the neonatal period, are a more likely cause of morbidity and mortality in NS. Such a pathogenic scenario implies that a severe disturbance in the cutaneous permeability barrier is an important feature of this subgroup of NS.

The normal skin barrier is provided by hydrophobic lipids organized into repeating arrays of lamellar membrane unit structures within the intercellular spaces of the stratum corneum (SC). In this study, we attempted to delineate a structural basis for the putative permeability barrier abnormality in NS using ruthenium tetroxide postfixation, which permits ultrastructural analysis of SC membrane structures,^18-20and to compare these findings with the ultrastructural characteristics of PsoE and CIE—disorders that clinically resemble the erythrodermic form of NS. Our findings suggest first, that premature lamellar body (LB) secretion is a distinctive feature of erythrodermic NS, and second, that severe SC extracellular abnormalities may account for the hypernatremic dehydration seen in severely affected patients.

In 6 of the 7 ultrastructurally studied NS cases, clinical data were available. They showed generalized involvement with a congenital or early-onset ichthyosiform erythroderma (Table 1 and Figure 1). Table 1 summarizes the sex, age, and clinical features of the patients at the time of investigations. In all patients except patient 3, diagnosis was ultimately established by demonstration of the typical hair shaft defect. All patients showed marked inflammation with areas of oozing, and in some, superficial blisters and erosions. Elliptical or punch biopsy specimens were taken from involved, uninfected skin sites.

Because of the close clinical resemblance of NS to PsoE and CIE, we also analyzed biopsy specimens from involved skin from 3 patients with PsoE and 2 with CIE. Patients with CIE fulfilled the following published criteria for autosomal recessive ichthyosis^21,22: collodion membrane at birth followed by subsequent erythroderma and fine scales in a generalized distribution and ectropion. Neither patient with CIE demonstrated genetic linkage to the transglutaminase: 1 (TGase1) gene or abnormal TGase1 expression.²³Finally, for morphologic control studies, we used freshly excised, surgical margins from 9 subjects without skin disease (from extensor surface; upper arms).

After rinsing in buffer, biopsy specimens were fixed in cacodylate-buffered 2.5% glutaraldehyde and then divided, with one half postfixed with 0.5% ruthenium tetroxide and 0.25% aqueous potassium ferrocyanide,^20,24and the other in 1% aqueous osmium tetroxide containing potassium ferrocyanide in the dark at 4°C. Tissue sections were dehydrated in graded ethanols and embedded in either ruthenium tetroxide–fixed samples (Spurr resin; Polysciences, Warrington, Pa) or osmium tetroxide–fixed samples (Epon 812; Polysciences). Thin sections were examined before and after double staining with ethanolic uranyl acetate plus lead citrate on an electron microscope operated at 100 kV. Serial semithin sections of the osmium tetroxide–fixed samples were stained with 1% methylene blue. Tissue specimens were also fixed and processed routinely for histologic testing, sectioned at 6 µm, and stained with hematoxylin-eosin, periodic acid-Schiff, and Giemsa staining.

Light microscopy of NS revealed psoriasiform features, with varying degrees of epidermal acanthosis and hyperkeratosis, accentuated rete ridges, and occasional long, narrow rete ridges (psoriasiform ichthyosis) (Table 2 and Figure 2, A).⁴ Marked parakeratosis—the absence or presence of only a partial granular layer—occurred commonly, whereas hypergranulosis was seen only focally in some patients. In 3 patients (2, 3, and 4), the SC was entirely parakeratotic. In all cases, the outermost nucleated cell layers did not flatten normally, instead showing irregularly distributed intracellular vacuolization and/or extracellular edema. Spongiosis was also pronounced in the lower epidermal cell layers. Focal accumulations of eosinophilic, periodic acid-Schiff–positive, diastase-resistant homogeneous material (Figure 2, B) occurred within the parakeratotic SC and focally within the stratum granulosum (SG) in only 1 patient (1).^5,16,25-29Finally, the papillary dermis showed a mild-to-marked perivascular inflammatory infiltrate consisting of histiocytes, lymphocytes, and some granulocytes, with regions of exocytosis.

Erythrodermic psoriasis was characterized by elongation of the epidermal rete ridges, with thickening of the deeper extensions and papillary edema. Thinning of the suprapapillary portion of the epidermis was also characteristic, with an occasional presence of small spongiform pustules. In some instances, the SC consisted entirely of parakeratotic cells, with a concomitant absence of the granular layer. Accumulations of pyknotic neutrophils were also present within parakeratotic areas of the SC (Munro microabscess).³⁰

The epidermis was slightly thickened, with broad rete ridges and a flattened base. The SC was thickened with foci of parakeratosis. In some regions, follicular hyperkeratosis was evident. The stratum granulation was expanded to 3 to 4 layers, and increased numbers of mitoses were visible in the basal cells. The dermis also showed a variable, patchy perivascular inflammatory infiltrate.²¹

In normal epidermis, exocytosis of LB contents occurred through fusion of the LB-limiting and plasma membranes, almost exclusively limited to the stratum granulosum (SG)/SC interface and subadjacent SG layer.^31,32 In the lowermost intercellular spaces of the SC, the secreted LB-derived lamellae began to uncoil within hemispherical, saccular dilatations at the SG-SC interface and lower SC (Figure 3, A).^24,31 The lateral margins of the unfurling lamellar membranes at this level seemed loosely connected to adjacent desmosomes.²⁰ Beneath the roof of the same extracellular domains, newly formed, mature lamellar membrane structures formed parallel arrays between neighboring desmosomes. The extracellular domains also displayed a uniform pattern of mature lamellar membrane structures throughout the mid- and upper SC (Figure 3, B).

Samples from NS displayed a number of features in common (Table 3). Cells in the outer nucleated layers of the epidermis appeared in various stages of transition into corneocytes. Moreover, even when transitional cells were not evident, the SC still appeared less cohesive than in normal SC (ie, they displayed fewer desmosomes and were often separated by elongated clefts) (Figure 4). Individual corneocytes showed numerous intracellular lipid droplets, nuclear remnants, and other inclusions (Figure 4, B).

Granular cells were not as flattened as normal, with irregularly distributed intracellular and/or extracellular edema. In edematous regions, keratohyalin granules and keratin filaments also appeared sparser than normal. Moreover, in patients 1, 2, 4, 5, and 7, keratohyalin granules lacked their characteristic stellatelike shape, and did not appear to interact normally with tonofibrils; patients 3 and 6 displayed an SG layered with 2 to 3 cells, with increased numbers of small, globular-shaped keratohyalin granules. Finally, in patients 1 and 2, spherical, dark cytoplasmic granules, with a diameter varying from 0.3 to 10 µm, were present in the SG cytosol (not shown).^7,26,27

The quantity of LBs in the SG varied greatly. In regions with severe cytosolic swelling, LBs were conspicuously reduced (Figure 4). In other regions, LBs displayed packed lamellar contents (Figure 5 and Figure 6, D), whereas in other regions, LBs revealed only lamellar fragments.

The intercellular spaces at the level of the stratum spinosum and SG were distended in some areas by amorphous, finely granular material (not shown).^3,33 In all patients except 1, this material also dilated the extracellular spaces of the overlying SG and SC. (In patient 1, the granular, intercellular deposits were limited to focal accumulations in the SC.) In some sites, the fine granular material formed a band that separated the SG layer from a parakeratotic SC.

In contrast to CIE and PsoE, LB secretion in NS occurred not only at the SG-SC interface, but also into the extracellular spaces of 4 or more layers of the subjacent SG and upper stratum spinosum (Figure 5 and Figure 6). The prematurely secreted lamellar contents remained unprocessed for up to 4 layers of the SC (Figure 5 and Figure 6, C and D). In some areas where the cornified envelope was already evident, elongated membrane sheets were present (Figure 6, A and B), but fully processed, mature lamellar membrane structures, such as in normal skin, did not occur (Figure 3, B). In extracellular domains of the lower SC, fusiform dilatations contained not only LB-derived sheets but also intermingled, electron-dense material, which persisted at and above sites where transformation into mature lamellar membrane structures occurred normally (Figure 7, B and C). The dilatated extracellular spaces of the mid-to-outer SC also displayed focal areas with normal membrane structures (patients 2, 4, and 5) separated by globular, electron-dense material (Figure 7, A), often in the vicinity of desmosomes.

The SC in PsoE was completely disorganized, showing increased numbers of parakeratotic corneocytes with remnants of nuclei and lipid droplets throughout. The extracellular spaces of the SC were unusually narrow, with only a few lamellar membranes evident,³⁴ and the mature pattern of lamellar membrane structures was not observed. Desmosomes appeared to be increased in some parts of the SC and elongated in others (Figure 8, C). In sites where neutrophils invaded the SC (Figure 8, A), the formation of intercellular dilatations, with deposition of abnormal lipid material, was seen (Figure 8, A). The LBs in the cytoplasm of the stratum spinosum and SG showed normal structures, with fusion of LBs and the cell membrane in the upper 2 layers of the SG, such as in normal human epidermis.³² Yet, the elongated LB-derived sheets persisted to higher layers within the SC interstices.³⁵ The cytosol of corneocytes also displayed numerous retained LB remnants.³⁶

As described,^34,37-39the SC in CIE revealed an irregular distribution of lamellar membranes in the extracellular spaces, with some regions containing excessive numbers of lamellar membranes that displayed an abnormal electron-lucent and electron-dense banding pattern. Extensive cleft formation also occurred between the lamellar membranes (Figure 8, D).

Furthermore, the surfaces of individual corneocytes appeared to be more undulated than normal (Figure 8, B), whereas the corneocyte matrix showed lipid droplets and clefts with longitudinal membrane structures.³⁷ Additionally, electron-lucent lacunae surrounded by electron-dense borders were found between the lamellar membranes (Figure 8, D). Although in some regions LBs showed only a few lamellar stacks or ovoid vesicles, other regions showed relatively normal-appearing LBs. Finally, in contrast to NS, extrusion of LB contents was limited to the upper SG and SG-SC interface.

The major function of the epidermis is to form a permeability barrier against excessive loss of bodily fluids. The permeability barrier resides in the SC and derives from the secretion of LB contents that reorganize to form organized arrays of hydrophobic membrane structures. Our ultrastructural study of erythrodermic NS demonstrated marked abnormalities of LB architecture, secretion, and membrane reorganization that likely signify a severe disturbance in permeability barrier function. The frequently observed hypernatremia and dehydration in NS could be explained by a defective barrier that resulted in increased loss of free water with reabsorption of solutes.⁵Our previous measurements of transepidermal water loss in NS (Table 1, patient 1) also showed a 4-fold increased rate of TEWL compared with a control infant of the same age.¹⁷A severe permeability barrier defect might also explain some unique therapeutic problems in NS, such as the propensity to develop iatrogenic Cushing syndrome⁴⁰ and aggravation of the dermatosis by retinoid therapy (which helps distinguish NS clinically from other ichthyosiform erythrodermas).⁶ In addition, failure to thrive in erythrodermic NS could also be ascribed, at least in part, to the barrier defect. A defective barrier would induce epidermal hyperplasia,⁴¹which, if severe and sustained, could induce a high catabolic state. As heat is lost through increased surface evaporation, energy requirements are increased.⁴² A defective barrier would increase the tendency to develop skin and systemic infections. Discomfort from pruritus and erosions⁶ could also further increase the caloric drain in these patients.

Although severe, recalcitrant hypernatremic dehydration is not uncommon in infantile erythrodermic NS, hypernatremia also occurs in other neonatal erythrodermas,⁴³ and therefore, this feature alone cannot be considered a specific clinical marker for NS. Moreover, erythroderma with failure to thrive may be the presenting feature of a number of disorders with diverse causes, including nutritional or metabolic disorders such as cystic fibrosis or acrodermatitis enteropathica (deficiency dermatitis)^44,45 and immunodeficiency syndromes.^15,46,47Therefore, features that could distinguish NS from other erythrodermic conditions, especially when there is a delayed appearance of bamboo hairs, could be crucial for the timely treatment of such infants.^5,6In this study, we attempted to identify diagnostically useful ultrastructural features of NS. To date, most ultrastructural studies have been within the context of isolated case reports of NS, either in the erythrodermic²⁷ or in the ILC^7,27-29phenotypes. Moreover, larger series^26,33failed to identify the distinctive features of NS vs other congenital erythrodermas. By applying the ruthenium tetroxide method in conjunction with osmium tetroxide postfixation, we identified certain ultrastructural alterations during the final stages of epidermal differentiation that could prove both diagnostically useful and functionally significant (Table 3).

First, secretion of LB contents occurs prematurely and is not followed by timely extracellular processing. In normal epidermis, extrusion of LB secretion occurs primarily at the SG-SC interface, followed sequentially by unfurling and elongation of LB-derived membrane sheets, which then transform into mature lamellar membrane unit structures in the lower SC.^19,20,31 In contrast, cornified envelope formation precedes the processing of LB sheets into mature extracellular lamellae by up to 4 SG layers in NS, a feature that is not observed in PsoE or CIE. Likewise, atopic dermatitis, a disorder that can resemble NS in neonates, shows delayed and incomplete LB secretion.³² Even at higher levels of the SC, extracellular processing of LB-derived sheets into mature lamellar membrane unit structures appears to be profoundly disturbed in NS. However, these features are not entirely specific because lesser but qualitatively similar processing abnormalities also occur in PsoE and CIE.

The extrusion of LB contents and their subsequent transformation are integrated within a tightly coordinated program of normal terminal differentiation,^45,48a sequence that appears to be profoundly disturbed in NS. The reduction in keratin and keratohyalin filaments in outer nucleated layers of the epidermis suggests that LB secretion and processing and expression of 1 or more structural proteins of epidermal terminal differentiation could be impaired in NS. Recently, a recessive mouse mutation with alopecia, abnormal hair (lanceolate hair), and thickening of the epidermis associated with an ichthyosiform dermatitis was described, and showed similarities to NS.⁴⁹Its mutation is located on the centromeric end of chromosome 18, a region homologous to human 18q12, and bears several candidate genes of epidermal differentiation, including cadherin, desmocollin, and desmoglein.⁴⁹ Abnormalities of proteins of the desmosomal plaque might result in defective interactions between cadherins and keratins (catenin-cadherin complexes are linked to the actin filament network and to other transmembrane and cytoplasmic proteins of the cytoskeleton⁵⁰). Yet, it is not clear how such a disturbance of cytoplasmic or desmosomal proteins could provoke the observed changes of LB secretion and lamellar membrane transformations described herein for NS. It has been speculated that desmosomes could play an important role during the early phase of lamellar membrane formation by either stabilizing and/or orientating the LB-derived sheets.²⁰

Prior studies have shown that extracellular calcium concentrations increase progressively from the basal to the outer SG layer and then decline^35,51 and that the epidermal calcium gradient regulates LB secretion.⁵² Thus, impaired formation of the epidermal calcium gradient in NS could account for premature LB secretion^32,51,53-55and inhibition of terminal differentiation. The calcium gradient is lost when the barrier is perturbed.⁵¹ Severity of the electrolyte abnormality in some patients with NS also suggests that the primary abnormality might involve ion channels or pumps. Regardless of the cause of the abnormal Ca++ gradient, a disturbed gradient could both accelerate the pathogenic features of NS after birth and account for the delayed onset of clinical and ultrastructural disease in some patients.³³

Second, transformation into mature lamellar membrane unit structures is disturbed by granular, electron-dense material. The severe barrier abnormality in NS can be explained by premature LB secretion and the extensive disruption of lamellar processing and organization observed herein. The amorphous, electron-dense material that forms clefts in NS may form an aqueous pore, perhaps explaining why barrier function is more impaired in NS than in other disorders of cornification where lipids are likely to form the abnormal, nonlamellar phase, eg, Refsum disease, Sjögren-Larsson syndrome,⁵⁶and neutral lipid storage disease.⁵⁷ The interaction of desmosomes (corneosomes) within the SC with lamellar membrane structures is also disturbed by the accumulation of excess electron-dense material in NS (Figure 6). This pathologic interaction might be responsible for the specific clinical feature of desquamation observed in ILC (double-edged scale).

In summary, the ultrastructural findings of the epidermal barrier components shown for NS are not observed in other congenital erythrodermic skin disorders (PsoE and CIE) that share clinical features in early infancy. We suggest, therefore, that the ultrastructural features described might assist in the early diagnosis of NS, leading to improved survival of these patients. Early diagnosis of NS vs other causes of erythrodermas is critical, because therapy differs widely. For example, systemic retinoids aggravate NS but can be useful for other erythrodermic conditions, such as CIE. Correct diagnosis also directs clinicians to carefully monitor both fluid and electrolyte status and caloric intake in these patients.

Accepted for publication February 26, 1999.

Corresponding author: Manigé Fartasch, MD, Department of Dermatology, University of Erlangen/Nuremberg, Hartmannstrasse 14, 91052 Erlangen, Germany (e-mail: fartasch@derma.med.uni-erlangen.de).