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Originally published as JHC exPRESS on September 20, 2005.
doi:10.1369/jhc.5A6701.2005
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Journal of Histochemistry and Cytochemistry
Volume 54 (1): 109-118, 2006
Copyright ©The Histochemical Society, Inc.

Colocalization of Multiple Laminin Isoforms Predominantly beneath Hemidesmosomes in the Upper Lamina Densa of the Epidermal Basement Membrane

James R. McMillan, Masashi Akiyama, Hideki Nakamura and Hiroshi Shimizu

Creative Research Initiative Sousei (JRM) and Department of Dermatology, Graduate School of Medicine (JRM,MA,HN,HS), Hokkaido University, Sapporo, Japan

Correspondence to: James R. McMillan, MSc, PhD, Department of Dermatology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, 060-8638, Japan. E-mail: jrm57{at}med.hokudai.ac.jp


   Summary
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 Summary
Introduction
 Materials and Methods
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 Literature Cited
 
Multiple laminin isoforms including laminins 5 ({alpha}3 ß3 {gamma}2), 6 ({alpha}3 ß1 {gamma}1), 10 ({alpha}5 ß1 {gamma}1), and possibly laminins 7 ({alpha}3 ß2 {gamma}1) and 11 ({alpha}5 ß2 {gamma}1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 ({alpha}3 ß3 {gamma}2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the {alpha}5, ß1, ß2, and {gamma}1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the {alpha}5, ß1, and {gamma}1 chains (present in laminins 10/11) and ß2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the {alpha}5, ß1, ß2, and {gamma}1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the {alpha}5, ß1, ß2, and {gamma}1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n≥200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the {alpha}5ß1/ß2{gamma}1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane. (J Histochem Cytochem 54:109–118, 2006)

Key Words: anchoring filament • epidermal basement membrane • hemidesmosome • immunoelectron microscopy • laminin 5 • laminin 10


   Introduction
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 Summary
 Introduction
Materials and Methods
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 Discussion
 Literature Cited
 
IN SKIN, laminins are present in the epidermal basement membrane, around blood vessels, nerves, and adnexal structures. It is generally thought that laminin 5 ({alpha}3 ß3 {gamma}2), possibly laminin 6 ({alpha}3 ß1 {gamma}1), and laminin 10 ({alpha}5 ß1 {gamma}1) are expressed in the human epidermal basement membrane (Aumailley and Rousselle 1999Go) (see Figure 1 ). The expression of laminins 7 ({alpha}3 ß2 {gamma}1) and 11 ({alpha}5 ß2 {gamma}1) has yet to be confirmed in the adult human epidermis (Aumailley and Rousselle 1999Go). Of these, laminin 5 ({alpha}3 ß3 {gamma}2) is the most well-studied epidermal isoform (Nishiyama et al. 2000Go; Mercurio et al. 2001Go; Geuijen and Sonnenberg 2002Go; McMillan et al. 2003bGo). Laminin 5 is thought to be crucial for the correct assembly and adhesion of hemidesmosomes (HDs) via the receptor, the {alpha}6ß4 integrin (Niessen et al. 1994Go).



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  		Figure 1

Schematic diagram showing the structure of laminin 5 and 10 chains and the position of two antibody binding sites in the {alpha}3 chain G1 domain (BM165) and the carboxyl terminal half of the {alpha}5 chain (4C7). This schematic diagram is not drawn to scale and does not include any {alpha}3 chain splice variants of laminin 5 ({alpha}3 ß3 {gamma}2). The antibody BM165 binds to the first globular (G1) domain of the {alpha}3 chain of laminins 5/6 (McMillan et al. 2003bGo), whereas the antibody 4C7 binds to the carboxyl terminal half end of the {alpha}5 chain (present in both laminins 10 and 11).

 
The distinct roles of laminin isoforms in the processes of cutaneous morphogenesis are poorly understood. Laminin 10 ({alpha}5 ß1 {gamma}1), however, has recently been implicated in several functions including hair follicle development (Li et al. 2003Go). In an {alpha}5 chain (laminin 10/11) knockout mouse model, the addition of exogenous laminin 10 was used to correct follicular development (Li et al. 2003Go). Laminin 10 is therefore implicated in hair follicle cell growth and adhesion (Gu et al. 2001Go; Pouliot et al. 2002Go; Li et al. 2003Go). Cell adhesion assays have demonstrated that multiple laminins (including laminins 5, 10, and 11) can act as adhesive substrates for keratinocytes and that this adhesion is mediated by the integrins {alpha}3ß1 and {alpha}6ß4 (Pouliot et al. 2002Go). However, in certain non-epithelial cells, the integrins {alpha}3ß1, {alpha}6ß1, and {alpha}6ß4 and {alpha} dystroglycan are expressed and have been identified as possible laminin 10/11 receptors (Kikkawa et al. 1998Go, 2000Go; Yu and Talts 2003Go).

Antibodies are now available that recognize specific laminin chains and provide new tools to investigate the structure of the epidermal basement membrane. These antibodies include 4C7 (Engvall et al. 1990Go; Tiger et al. 1997Go) that recognizes a carboxyl terminal domain of the human {alpha}5 chain of laminins 10 ({alpha}5 ß1 {gamma}1, see Figure 1) and 11 ({alpha}5 ß2 {gamma}1). This antibody blocks the epitope involved in neurite cell adhesion to the laminin {alpha}5 chain (Engvall et al. 1986Go; Makino et al. 2002Go). Further laminin-specific antibodies to ß and {gamma} chains include 2E8 (recognizing the ß1 chain (Engvall et al. 1986Go), D18 ({gamma}1) (Sanes et al. 1990Go), and C4 (ß2) (Hunter et al. 1989Go).

To better understand the position and possible functions of epidermal molecules, we examined the precise localization of the {alpha}5, ß1, ß2, and {gamma}1 laminin chains and collagen IV in the interfollicular and follicular epidermal basement membrane. In addition, the expression of laminin chains was also assessed in a range of epidermolysis bullosa (EB) patients' skin harboring defects in several basement membrane components, including laminin 5. We have quantitatively analyzed and compared the localizations of laminin {alpha}5, ß1, ß2, and {gamma}1 chains with that of laminin 5 ({alpha}3 ß3 {gamma}2) from our previously published data (McMillan et al. 2003bGo) and collagen IV. Comparison of {alpha}5, ß1, ß2, and {gamma}1 chain expression with laminin 5, a well-studied HD-associated isoform, will determine a more precise localization for these laminin isoforms. Our data support the hypothesis that multiple laminin isoforms colocalize beneath HDs in normal and diseased epidermal basement membranes.


   Materials and Methods
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 Summary
 Introduction
 Materials and Methods
Results
 Discussion
 Literature Cited
 
Skin Samples
Samples of adult and neonatal control skin from non-spe cialized sites (abdomen, arm, thigh, n=8; and scalp skin, n=2) were obtained from routine surgical procedures. Skin samples were frozen for cryostat sectioning or processed for postembedding immunogold electron microscopy (IEM) as described below. In all cases, the biopsies were performed with the patient's or guardian's informed consent, with the relevant institutional approval for experiments handling human material, and in accordance with the Helsinki Declaration.

Skin samples from patients affected with a group of rare genodermatoses, EB, were included in this study (n=15, see Table 1). Details of the number of patients for each EB disease subtype, their age at biopsy, details of any identified mutations, or significant results of diagnostic antibody staining are listed, in addition to the results of their laminin antibody staining findings (see Table 1). Four Herlitz junctional (HJ) EB patients harbored laminin-5 chain mutations that were reported in the literature (Takizawa et al. 1998aGoGoGo–dGo). In one EB simplex associated with muscular dystrophy (EBS-MD) patient, genetic defects have been reported (Pulkkinen et al. 1996Go).


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  		Table 1

Comparison of laminin 5 and laminin 10 expression in patients with various forms of epidermolysis bullosa

 
Confocal Immunofluorescence Microscopy
Indirect immunofluorescence was performed as previously described (Kennedy et al. 1985Go) using cryostat skin sections. Laminin chain expression was confirmed in control skin using the following antibodies: 4C7 recognizing the human {alpha}5 chain (see Figure 1) present in laminins 10 and 11 (dilution 1:25; Chemicon International, Temecula, CA) (Engvall et al. 1990Go; Tiger et al. 1997Go). The monoclonal antibody 2E8 recognizing the ß1 chain (neat) (Engvall et al. 1986Go), the monoclonal antibody D18 that recognizes the {gamma}1 chain (see Figure 1) (neat) (Sanes et al. 1990Go), and an antibody C4 to the ß2 chain (see Figure 1) (used neat) (Hunter et al. 1989Go) were also included. The antibodies 2E8, D18, and C4 were obtained from the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA). The mouse monoclonal M3F7 recognizing the helical domain of the {alpha}1 and {alpha}2 chains of collagen IV (used neat) (Foellmer et al. 1983Go) was also obtained from the Developmental Studies Hybridoma Bank. Laminin-5 antibodies included the mouse monoclonal BM165 directed against the laminin-5 {alpha}3 chain terminal first globular (G1) domain (see Figure 1) (diluted 1:50) (Marinkovich MP, unpublished data) (McMillan et al. 2003bGo); K140 directed against the laminin-5 ß3 chain adjacent to domain IV; GB3 directed against the laminin-5 {gamma}2 chain (Harlan Sera Lab; Loughborough, UK); and a rabbit polyclonal serum directed against the entire laminin-5 molecule (1:200) (McMillan et al. 2003bGo). The melanocyte marker antibody TMH-1 recognized the b-locus protein (rat antibody, 1:10 dilution) and was previously described by Masunaga et al. (1996)Go.

Epidermal sections were fixed in cold acetone (–20C) for 10 min and incubated with 5% normal rabbit sera in 0.1 M Dulbecco's PBS for 5 min at 37C. Sections were incubated with primary antibodies and subsequently with secondary antibodies conjugated to fluorescein isothiocyanate or Texas Red (FITC; rabbit anti-mouse IgG or goat anti-rabbit IgG, 1:200; DAKO, Tokyo, Japan; Texas Red conjugated donkey anti-rabbit; Amersham, UK). To label TMH-1, a preabsorbed cyanine (CY5)-conjugated goat anti-rat antibody was used (Jackson ImmunoResearch; West Grove, PA). All secondary antibodies were diluted in 3% BSA in 0.1 M PBS for 30 min at 37C in a darkened, humidified chamber. Sections were then labeled with a ToPro-3 nuclear counterstain (diluted 1:20,000, blue channel; Jackson ImmunoResearch) if appropriate. The sections were then mounted in Permafluor (Thermo Shandon; Pittsburgh, PA) and examined with a confocal microscope (Fluoview FV300; Olympus, Tokyo, Japan) using an inverted microscope (IX70; Olympus). Controls included normal skin cryostat sections with the primary antibody substituted by PBS, myeloma supernatant, or an irrelevant immunoglobulin isotype, as a negative control. All experiments were performed at least in duplicate.

Immunogold Electron Microscopy
Four samples of human skin were cryofixed and processed for postembedding IEM according to the previously described methods (Shimizu et al. 1989Go,1990Go). Samples were washed in PBS and cryoprotected in 20% glycerol (in PBS) for up to 1 hr at 4C. Subsequently, cryofixation was performed in liquid propane at –190C using a freeze plunge apparatus (Leica CPC; Cambridge, UK) followed by freeze substitution over 3 days at –80C in methanol using an automated freeze substitution system (AFS; Leica). Specimens were embedded in Lowicryl K11M (Ladd Research Industries; Burlington, VT) resin over 4 days at –60C. The temperature was gradually raised and the resin was polymerized under UV light and liquid nitrogen vapor at 10C. Ultrathin sections were then cut and collected on pioloform-coated nickel grids. Sections were stained with uranyl acetate only (15 min) and observed with a transmission electron microscope (Hitachi H-7100; Tokyo, Japan) at 75 kV. Blocks showing good ultrastructure were selected for immunolabeling experiments. Sections were preincubated in buffer containing PBS with 5% normal goat serum (NGS), 1% BSA, and 0.1% gelatin. Primary antibodies or human antisera were all diluted in PBS buffer containing 1% NGS, 1% BSA, and 0.1% gelatin and incubated at 37C for 2 hr. The sections were then washed in a drop of PBS buffer four times (5 min each) and placed on a drop of secondary linker antibody, again diluted in PBS buffer (for 2 hr at 37C). The secondary antiserum, rabbit anti-mouse IgG (DAKO; Ely, UK) was diluted 1:500. Sections were then incubated with a final antibody layer using 5-nm gold-conjugated labeled goat anti-rabbit or goat anti-mouse antibodies (Biocell; Cardiff, UK) diluted 1:500 in Tris-buffered saline (TBS) for 2 hr at 37C. For double labeling on K11M sections, the {alpha}5 chain of laminins 10/11 (4C7 and a 5-nm gold-conjugated goat anti-mouse) and rabbit anti-laminin 5 (polyclonal highlighted by 15-nm gold-conjugated goat anti-rabbit; Biocell) were used. Sections were washed twice in TBS buffer and twice in distilled water (5 min each). After staining with 15% alcoholic uranyl acetate (3 min) and lead citrate (15 min), sections were observed with a transmission electron microscope (H-7100; Hitachi). Controls included normal skin sections with the primary antibody substituted by PBS, myeloma supernatant, preimmune rabbit serum, or an irrelevant immunoglobulin isotype, as a negative control. All experiments were performed in triplicate.

Immunogold Quantitative Analysis
The techniques for ultrastructural labeling were similar to those performed by McMillan et al. (2003b)Go. Electron micrographs were taken at a standard magnification (30K) and were enlarged by a standard factor x2.08. The final magnification (x62,500) was checked using electron micrographs taken of a carbon diffraction grating. For standardization purposes, all observations were made by one observer (JRM). At least 200 gold particles were assessed per specimen for each antibody or antiserum and four specimens from different individuals were examined (see Table 1 and Table 2). A 5-nm immunogold-conjugated final antibody layer was used. The percentage of gold particles perpendicularly beneath an observable electron-dense HD cytoplasmic outer attachment plaque as described by McMillan and Eady (1996)Go was scored and calculated from a large number of gold particles in skin from four individuals.


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  		Table 2

Immunogold particle distribution shows the majority of laminin labeling is restricted to beneath the hemidesmosome (HD) plaque

 
Only non-obliquely sectioned areas of dermal–epidermal junction were included with clearly defined HD plaques, lamina lucida (LL), and lamina densa (LD). The dermal–epidermal junction beneath melanocytes or in damaged areas was excluded from this study. Gold particles that appeared clumped or associated with any deposit were excluded.

For each antibody or antisera, the positions of gold particles were statistically tested by one-way ANOVA and a two-sample t-test using the Minitab statistical package (Minitab Inc; University of Pennsylvania, Philadelphia, PA). An antibody (4C7) that recognizes a carboxyl terminal domain of the {alpha}5 chain of laminins 10/11 was used to determine the mean position of labeling directly beneath the keratinocyte plasma membrane (Engvall et al. 1986Go; Makino et al. 2002Go)(see Figure 1 for epitope position). The labeling of the {alpha}5 chain was compared with the distribution of the G1 domain of laminin-5 {alpha}3 chain (using data previously reported by McMillan et al. 2003bGo).


   Results
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 Summary
 Introduction
 Materials and Methods
 Results
Discussion
 Literature Cited
 
Confocal Fluorescence Microscopy of Control Skin
Laminin-5 staining was restricted to the dermal–epidermal junction in control skin (data not shown). This was similar to the dermal–epidermal junction staining of {alpha}5 chain of laminins 10 (data not shown). Laminins 10 and 11 were also expressed in dermal blood vessels. Laminin 11 (as identified by the ß2 chain) dermal–epidermal junction staining was present in adult control thigh and arm skin but was variable in other samples including scalp skin. Therefore, ß2 chain expression appears to be distinct and independent from that of the {alpha}5 chain. Staining for the {alpha}5, ß1, and {gamma}1 chains was weaker in the adult dermal–epidermal junction than in blood vessels (data not shown), whereas dermal–epidermal junction staining was generally brighter in younger skin samples (<16 years, data not shown). This would appear to support a previous report of age-dependent expression of the laminin 10/11 chains (Pouliot et al. 2002Go).

Confocal fluorescence microscopy (Figures 2A –2C) showed that both laminin-5 and {alpha}5 chains are expressed in the dermal–epidermal junction of control interfollicular epidermis except for small gaps (white arrows in Figures 2A and 2B) beneath small isolated cells presumed to be melanocytes staining blue for the melanocyte marker, TMH-1 (Figures 2A and 2B) (Masunaga et al. 1996Go). These data suggest that laminin-10 chains are restricted to beneath keratinocytes and are not expressed beneath melanocytes.



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  		Figure 2

Reduced laminin 10 (green, FITC) and laminin 5 (red, Texas Red) labeling below the melanocyte marker TMH-1 (blue, CY-5) of normal adult control skin. Both laminin 5 and the {alpha}5 chain colocalize (orange color) within the dermal–epidermal junction (A) and both are expressed only weakly or are absent beneath melanocytes (B,C, in blue, see white arrows). The {alpha}5 chain of laminins 10/11 together with laminin 5 is not expressed beneath melanocytes (A–C, white arrows) but does show a distinct, strong expression pattern that includes dermal vessels (A–C, open arrows). Bar = 25 µm; Inset C = 10 µm.

 
A previous scalp skin and hair follicle immunohistochemical study (Akiyama et al. 1995Go) demonstrated a specific staining pattern for many HD and anchoring filament components, particularly laminin 5, which manifests as reduced staining around the lower hair bulb and a reemergence of staining over the dermal papilla region (Akiyama et al. 1995Go). We observed this characteristic pattern of staining along the majority of the follicles, for both laminin 5 (bracketed area in Figure 3A ) and the laminin {alpha}5 chain (brackets and dotted line in Figures 3B and 3D). The laminin ß1 and {gamma}1 chains also showed this staining pattern (data not shown). There was no staining for the ß2 chain in the hair follicle (data not shown). The dermal–epidermal junction of the bulge region stained for both {alpha}3 and the {alpha}5 chains (Figures 3A and 3B, Bu) and the {alpha}5 chain also stained the erector pili muscle (Figure 3C, EP).



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  		Figure 3

Indirect immunofluorescence shows a typical hemidesmosomal (HD) component-like expression pattern of the laminin {alpha}5 chain in late anagen human hair follicles. A previous scalp hair follicle immunohistochemical study (Akiyama et al. 1995Go) demonstrated a specific staining pattern for many HD-anchoring filament-associated components including laminin 5. In our study, both laminin-5 polyclonal staining (A) and the laminin {alpha}5 chain (B, 4C7) showed characteristic bright patterns along the epidermal proximal hair shaft including the bulge region (B,C, Bu) but progressively weaker staining toward the hair bulb (D, bracketed areas) with staining becoming brighter again higher up the hair shaft. Laminin {alpha}5 chain staining was present in the shaft (B) bulge region (B,C) and the apical tip of the hair bulb matrix (D). Laminin ß1 and {gamma}1 chains showed similar staining to the {alpha}5 chain (data not shown). No laminin ß2 chain staining was detected in the hair follicle (data not shown). Bar = 25 µm.

 
Confocal Fluorescence Microscopy of Epidermolysis Bullosa Skin
The expression of laminins 5 ({gamma}2 chain), 10 (all chains), and 11 (ß2 chain) in patients with different forms of EB was compared. In both control (Figure 4A ) and all of the EB subtypes (Figures 4B–4H), {alpha}5, ß1, ß2, and {gamma}1 chain expression was detectable. Laminin expression was weak in areas of split skin particularly in HJEB with defects in laminin 5 ({alpha}5 chain, Figures 4B and 4C, respectively, asterisks show the split area) (Uitto and Pulkkinen 2001Go). The reduction in {alpha}5 and ß2 chain expression in HJEB patients, particularly over split skin, suggested that this effect might be due to antigen degradation in vivo in the split areas. The presence of {alpha}5 and ß2 chains in intact EB skin, however, confirms that these chains are capable of being independently synthesized and assembled even in the total absence or in the presence of defective laminin 5. All other EB cases also showed normal staining for other laminins including junctional EB associated with pyloric atresia (JEB-PA) (with defects in {alpha}6ß4 integrin, Figure 4E), EB simplex associated with muscular dystrophy (EBS-MD) (with defects in plectin, Figure 4F), non-lethal junctional (NLJEB) (with defects in BP180, Figure 4G), and severe recessive dystrophic epidermolysis bullosa (SRDEB) (Figure 4H). In control and SRDEB patients' skin there was normal staining for laminin 5 ({gamma}2 chain using the antibody GB3, see Figure 4L). This was in contrast to laminin-5 chain staining that was severely reduced or absent in both the NLJEB (Figure 4J) and HJEB cases (Figure 4K), harboring severe defects in laminin-5 expression.



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  		Figure 4

Laminin {alpha}5 and ß2 chains are expressed beneath the epidermis in epidermolysis bullosa (EB) skin but are focally reduced in split lethal Herlitz junctional EB (HJEB) skin. The laminin {alpha}5 chain in control skin showed linear fluorescence along the dermal–epidermal junction and in dermal blood vessels (A). However, in non-lethal (B) and HJEB skin (C,D), the laminin {alpha}5 and ß2 chains showed reductions in dermal–epidermal junction staining, especially where areas of skin had become separated (C, asterisks). This effect was due to antigen degradation in HJEB skin during skin separation as demonstrated by reduced collagen IV staining (O, arrows:) over the split areas (O, asterisks), whereas control skin (M), non-lethal junctional EB (NLJEB) (N) and dystrophic EB skin (P) showed bright linear collagen IV staining, respectively. The presence of {alpha}5 and ß2 chain staining in intact EB skin demonstrates that these chains are independently synthesized and maintained even in the presence of other defective basement membrane components. All other cases of EB showed normal staining for the laminin {alpha}5 chain (and ß2 chain, not shown), including junctional EB associated with pyloric atresia (JEB-PA with defects in {alpha}6ß4 integrin (E), EB simplex associated with muscular dystrophy (EBS-MD with defects in plectin) (F), NLJEBBP180 with defects in BP180 (G), and severe recessive dystrophic EB (SRDEB with collagen VII defects (H). In control and SRDEB patients' skin, laminin-5 {gamma}2 chains were normally expressed (I and L, respectively) using the {gamma}2 chain monoclonal antibody GB3. Laminin-5 expression was severely reduced and absent in both the NLJEB and HJEB cases (J,K), respectively, harboring severe defects in laminin 5. Bar = 50 µm.

 
Immunogold Electron Microscopy and Quantitative Analysis
Labeling of control interfollicular epidermal sections showed that the majority of laminin-10 {alpha}5 chains (Figures 5A –5D; Table 2), ß1, {gamma}1, and 11 chain (ß2 chain, data not shown) were restricted to under the cytoplasmic HD outer plaques. This was in contrast to collagen IV, which was not as restricted to beneath epidermal HDs plaques (see Figure 5F, 61% collagen IV vs 82% laminin 5). The difference between all laminin and collagen values was statistically significant using the one-way ANOVA (p<0.001) and Student's t-test (p<0.000). All four {alpha}5, ß1, ß2, and {gamma}1 chain antibodies and antiserum showed a remarkable similarity in the percentage of labeling associated with the HD attachment plaque and anchoring filament complex beneath HDs, ranging between 84% and 92% (see Table 2). Furthermore, these values reflect an almost identical (HD restricted) expression pattern to the previously reported values for laminin-5 subunits (Masunaga et al. 1996Go; McMillan et al. 2003bGo) (part of these findings are also included in Table 2). Our data are very similar to those of laminin 5 ({alpha}3 chain) that on average demonstrated 82% of labeling restricted to beneath HDs (see Table 2) at a distance ranging from 35 to 45 nm below the plasma membrane at the LL–LD junction (Masunaga et al. 1996Go; McMillan et al. 2003bGo). The precise distance of the 4C7 epitope on the C-terminal portion of the {alpha}5 chain was 53.07 nm (±6.69 SD) from the plasma membrane (arrows, Figures 5A– 5D). This is 18 nm lower than the G1 domain of the laminin-5 {alpha}3 chain described previously (see Figure 1) (McMillan et al. 2003bGo). The difference between these two {alpha} chain mean values was statistically significant using the one-way ANOVA (p>0.01) and Student's t-test (p=0.009). However, visual examination of the distribution of these two antigens revealed overlapping values ~30–40 nm beneath the plasma membrane, the only difference being that the {alpha}5 chain showed a wider range of labeling that extended deeper in the LD compared with the {alpha}3 chain. The remaining three ß1-, ß2-, and {gamma}1-chain antibodies recognized, as yet unidentified, epitopes on specific laminin chains and were therefore not included in the plasma membrane distance measurements. However, all three antibodies showed upper LD labeling (not shown), the majority of which were restricted to beneath HDs similar to the {alpha}5 chain. The three ß1-, ß2-, and {gamma}1-chain antibodies were excluded from the distance measurements but were scored for their localization either beneath visible HD attachment plaques (as defined by McMillan and Eady 1996Go) or within inter-HD areas.



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  		Figure 5

The majority of laminin 5 and laminin 10/11 chains are restricted to the lamina densa beneath HDs, whereas collagen IV is expressed continuously along the basal lamina. Postembedding immunoelectron microscopy with anti-{alpha}5 chain (laminin 10) (A–D) and double labeling with anti-{alpha}3 chain (laminin 5) (E) antibodies in control skin reveals a similar labeling pattern for these laminins. Collagen IV (M3F7), however, is not restricted to beneath HDs but is continuous along the basal lamina (F). Double labeling with laminin 5 polyclonal antiserum (15 nm gold particles) and laminin 10/11 (with smaller 5 nm gold particles) shows that the majority of labeling colocalizes beneath HDs at the border between the lamina lucida and lamina densa (E). The majority (84%) of laminin {alpha}5 chain labeling (see Table 2 and A–D) was restricted to areas immediately beneath the HDs, over the LD border (A–D, arrows) whereas only 61% of collagen IV was restricted to beneath HDs (F). HD plaques (solid arrows) are shown within the keratinocyte cytoplasm (white cross) and the lamina lucida highlighted by asterisks. Bar = 0.2 µm.

 
Double labeling for the {alpha}5 chain of laminins 10/11 (highlighted by 5-nm small gold particles) and whole anti-laminin 5 antiserum (shown by the larger 15-nm gold particles) shows a similar labeling pattern in the LD beneath electron densities presumed to be HDs (Figure 5E). HD plaques are visible within the keratinocyte cytoplasm (white cross) and the dermal–epidermal junction is separated by the LL (Figure 5E, asterisks). Together our data suggest that the {alpha}5 chains (including ß1, ß2, and {gamma}1 chains, see Table 2) show a restricted expression pattern beneath HDs, similar to laminin 5 but unlike collagen IV.


   Discussion
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
Literature Cited
 
We have demonstrated that the {alpha}5, ß1, and {gamma}1 chains show a similar localization to laminin 5 in the human interfollicular epidermal basement membrane. These data support the presence of multiple laminin isoforms beneath HDs in the basement membrane at several different epidermal sites. A very different localization of collagen IV within the LD but not restricted to beneath HDs was observed. These data suggest a complex network of interactions between different basement membrane components beneath the epidermis (Ghohestani et al. 2001Go; McMillan et al. 2003aGo; Miner and Yurchenco 2004Go).

In addition we have demonstrated that the expression of the {alpha}5 and ß2 chains is independent of laminin 5, as demonstrated by residual staining in HJEB patients' skin. The reduction in {alpha}5 and ß2 chain expression in HJEB was only observed over separated, blistered areas of skin, suggesting that this effect is due to separation-induced antigen degradation in vivo. This was supported by reduced collagen IV staining in split areas of EB skin. This was confirmed after reduced collagen IV staining was observed within separated areas of HJEB skin samples (data not shown). Our results also suggest that the presence of other laminins cannot fully compensate for defects in laminin-5-deficient HJEB skin (McMillan et al. 1997Go,1998Go). The {alpha}5 and ß2 chains were also normally expressed in all other EB samples harboring defects in plectin, collagen XVII (bullous pemphigoid antigen 2), the {alpha}6ß4 integrin, and collagen VII.

In previous reports (Aumailley and Rousselle 1999Go), the laminin ß2 chain was not expressed in the epidermal basement membrane of neonatal foreskin. However, we showed weak, variable ß2 chain expression (in thigh and arm skin) and absences in other body sites (scalp skin). We conclude that the lack of ß2 staining may be due to several factors: antigen masking, a low-level expression, or site- or age-specific variations in human laminin ß2 chain expression that may include posttranslational protein processing seen in other laminin isoforms (Miner et al. 1997Go).

Laminin 5, together with several HD-associated antigens, is expressed in a specific pattern around late anagen hair follicles that excludes staining around the dermal papilla area (Akiyama et al. 1995Go; Nutbrown and Randall 1995Go). Laminin-10 chains also show this similar expression pattern in late anagen hair follicles. Unlike laminin-5 and laminin-10 chains, we failed to observe any ß2 chain expression (laminins 7/11) in any part of the adult hair follicle; however, this may be due to a low level of antigen expression or masking of the ß2 chain epitope. The significance of these findings may be related to the specific growth phases of the lower non-permanent portion of the hair follicle.

In the laminin {alpha}5 chain knockout mouse, an unusual disruption in hair follicle morphogenesis was demonstrated (Li, et al., 2003). Li et al. (2003)Go reported that, in control mice, laminin 10 was present in murine elongating hair germs when other laminins were downregulated, suggesting a specific role for this laminin in hair follicle development and follicular keratinocyte migration. Mouse skin lacking laminin 10 also contained fewer hair germs and follicles compared with control mice, and after transplantation experiments this skin showed a failure of hair germ elongation and defective basement membrane assembly. Intriguingly, treatment of these mice with purified exogenous laminin 10 corrected these defects and restored hair follicle development. Given that human hair follicles are slow cycling and the majority remains in the late anagen phase and shows different growth characteristics to murine follicles, our failure to demonstrate such growth phase-specific differences in the expression of laminin 10 during the hair cycle stages is not surprising.

The presence of multiple laminin isoforms beneath HDs suggests the hypothesis that there are laminin subunits possibly with overlapping functions that form focal clusters of laminin molecules. This was in contrast to collagen IV, which was not restricted to HDs and localized to the LD region. Ultrastructural data show that the {alpha}3 chain (laminin 5) is closer to the plasma membrane than the {alpha}5 chain (with only an 18-nm difference, see Table 2). This might suggest that the {alpha}5 chain is more closely associated with a LD component such as collagen IV. However, given the size of both laminin chains of ~80–100 nm (as determined by rotary shadowing experiments), our data suggest a significant overlap occurs between {alpha}3 and {alpha}5 chains (Marinkovich et al. 1992Go; Vailly et al. 1994Go). Further studies using a larger battery of antibodies are required to determine the orientation of these laminin components.

Together these data show for the first time that laminin 10/11 chains are restricted to beneath HDs similar to laminin 5 but distinct from collagen IV. Our data suggest a specific localization of multiple laminin isoforms in the epidermal basement membrane beneath HDs and support the hypothesis that several laminins in close association may promote stable cell attachment among different basement membrane molecules.


   Acknowledgments
 
This work was supported by a grant-in-aid of Scientific Research A (13357008, HS) and Health and Labor Sciences Research Grants (Research into Specific Diseases) H13-Saisei-02 and H17-Saisei 12, by a grant from the Japanese Society for the Promotion of Science (JSPS) grant #00345 to J.R.M., and by a grant-in-aid for JSPS fellows' research expenses (#00345). This work was also supported by a grant from the Japanese Health Science Foundation for Research Residents, for class "A" researchers (JRM).

We gratefully acknowledge the technical support of Ms. M. Sato and Ms. K. Sakai in this study. We also thank Dr. T. Masunaga for kindly providing the data from the {gamma}2 chain study (Masunaga et al. 1996Go) and Dr. M.P. Marinkovich for his kind gift of his polyclonal laminin 5 antibody. Hybridoma supernatants D18 and 2E8 (produced by Drs. J. Sanes and E. Engvall) and M3F7 (from Dr. H. Furthmeyer) were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development (NICHD) and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa.


   Footnotes
 
Received for publication March 28, 2005; accepted September 6, 2005


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Akiyama M, Dale BA, Sun TT, Holbrook KA (1995) Characterization of hair follicle bulge in human fetal skin: the human fetal bulge is a pool of undifferentiated keratinocytes. J Invest Dermatol 105: 844–850[CrossRef][Medline]

Aumailley M, Rousselle P (1999) Laminins of the dermo-epidermal junction. Matrix Biol 18:19–28[CrossRef][Medline]

Engvall E, Davis GE, Dickerson K, Ruoslahti E, Varon S, Manthorpe M (1986) Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol 103:2457–2465[Abstract/Free Full Text]

Engvall E, Earwicker D, Haaparanta T, Ruoslahti E, Sanes JR (1990) Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul 1:731–740[Medline]

Foellmer HG, Madri JA, Furthmayr H (1983) Methods in laboratory investigation. Monoclonal antibodies to type IV collagen: probes for the study of structure and function of basement membranes. Lab Invest 48:639–649[Medline]

Geuijen CA, Sonnenberg A (2002) Dynamics of the {alpha}6ß4 integrin in keratinocytes. Mol Biol Cell 13:3845–3858[Abstract/Free Full Text]

Ghohestani RF, Li K, Rousselle P, Uitto J (2001) Molecular organization of the cutaneous basement membrane zone. Clin Dermatol 19:551–562[CrossRef][Medline]

Gu J, Sumida Y, Sanzen N, Sekiguchi K (2001) Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130(Cas)-CrkII-DOCK180 pathway. J Biol Chem 276:27090–27097[Abstract/Free Full Text]

Hunter DD, Shah V, Merlie JP, Sanes JR (1989) A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature 338:229–234[CrossRef][Medline]

Kennedy AR, Heagerty AH, Ortonne JP, Hsi BL, Yeh CJ, Eady RA (1985) Abnormal binding of an anti-amnion antibody to epidermal basement membrane provides a novel diagnostic probe for junctional epidermolysis bullosa. Br J Dermatol 113:651–659[CrossRef][Medline]

Kikkawa Y, Sanzen N, Fujiwara H, Sonnenberg A, Sekiguchi K (2000) Integrin binding specificity of laminin-10/11: laminin-10/11 are recognized by {alpha}3ß1, {alpha}6ß1 and {alpha}6ß4 integrins. J Cell Sci 113:869–876[Abstract]

Kikkawa Y, Sanzen N, Sekiguchi K (1998) Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells. Laminin-10/11 mediates cell adhesion through integrin {alpha}3ß1. J Biol Chem 273:15854–15859[Abstract/Free Full Text]

Li J, Tzu J, Chen Y, Zhang YP, Nguyen NT, Gao J, Bradley M, et al. (2003) Laminin-10 is crucial for hair morphogenesis. EMBO J 22: 2400–2410[CrossRef][Medline]

Makino M, Okazaki I, Kasai S, Nishi N, Bougaeva M, Weeks BS, Otaka A, et al. (2002) Identification of cell binding sites in the laminin {alpha}5-chain G domain. Exp Cell Res 277:95–106[CrossRef][Medline]

Marinkovich MP, Lunstrum GP, Burgeson RE (1992) The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 267:17900–17906[Abstract/Free Full Text]

Masunaga T, Shimizu H, Ishiko A, Tomita Y, Aberdam D, Ortonne JP, Nishikawa T (1996) Localization of laminin-5 in the epidermal basement membrane. J Histochem Cytochem 44:1223–1230[Abstract]

McMillan JR, Akiyama M, Shimizu H (2003a) Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions. J Dermatol Sci 31:169–177[CrossRef][Medline]

McMillan JR, Akiyama M, Shimizu H (2003b) Ultrastructural orientation of laminin 5 in the epidermal basement membrane: an updated model for basement membrane organization. J Histochem Cytochem 51:1299–1306[Abstract/Free Full Text]

McMillan JR, Eady RA (1996) Hemidesmosome ontogeny in digit skin of the human fetus. Arch Dermatol Res 288:91–97[Medline]

McMillan JR, McGrath JA, Pulkkinen L, Kon A, Burgeson RE, Ortonne J-P, Meneguzzi G, et al. (1997) Immunohistochemical analysis of skin in junctional epidermolysis bullosa using laminin 5 chain specific antibodies is of limited value in predicting the underlying gene mutation. Br J Dermatol 136:817–822

McMillan JR, McGrath JA, Tidman MJ, Eady RA (1998) Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa. J Invest Dermatol 110:132–137[CrossRef][Medline]

Mercurio AM, Rabinovitz I, Shaw LM (2001) The {alpha}6ß4 integrin and epithelial cell migration. Curr Opin Cell Biol 13:541–545[CrossRef][Medline]

Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, et al. (1997) The laminin {alpha} chains: expression, developmental transitions, and chromosomal locations of {alpha}1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel {alpha}3 isoform. J Cell Biol 137:685–701

Miner JH, Yurchenco PD (2004) Laminin functions in tissue morphogenesis. Annu Rev Cell Dev Biol 20:255–284[CrossRef][Medline]

Niessen CM, Hogervorst F, Jaspars LH, de Melker AA, Delwel GO, Hulsman EH, Kuikman I, et al. (1994) The {alpha}6ß4 integrin is a receptor for both laminin and kalinin. Exp Cell Res 211:360–367[CrossRef][Medline]

Nishiyama T, Amano S, Tsunenaga M, Kadoya K, Takeda A, Adachi E, Burgeson RE (2000) The importance of laminin 5 in the dermal-epidermal basement membrane. J Dermatol Sci 24(suppl 1): S51–59

Nutbrown M, Randall VA (1995) Differences between connective tissue-epithelial junctions in human skin and the anagen hair follicle. J Invest Dermatol 104:90–94[CrossRef][Medline]

Pouliot N, Saunders NA, Kaur P (2002) Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration. Exp Dermatol 11: 387–397[CrossRef][Medline]

Pulkkinen L, Smith FJ, Shimizu H, Murata S, Yaoita H, Hachisuka H, Nishikawa T, et al. (1996) Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy. Hum Mol Genet 5:1539–1546[Abstract/Free Full Text]

Sanes JR, Engvall E, Butkowski R, Hunter DD (1990) Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol 111: 1685–1699[Abstract/Free Full Text]

Shimizu H, McDonald JN, Gunner DB, Black MM, Bhogal B, Leigh IM, Whitehead PC, et al. (1990) Epidermolysis bullosa acquisita antigen and the carboxy terminus of type VII collagen have a common immunolocalization to anchoring fibrils and lamina densa of basement membrane. Br J Dermatol 122:577–585[CrossRef][Medline]

Shimizu H, McDonald JN, Kennedy AR, Eady RAJ (1989) Demonstration of intra- and extra-cellular localization of bullous pemphigoid antigen using cryofixation and freeze substitution for postembedding immuno-electron microscopy. Arch Dermatol Res 281:443–448[CrossRef][Medline]

Takizawa Y, Pulkkinen L, Shimizu H, Lin L, Hagiwara S, Nishikawa T, Uitto J (1998a) Maternal uniparental meroisodisomy in the LAMB3 region of chromosome 1 results in lethal junctional epidermolysis bullosa. J Invest Dermatol 110:828–831[CrossRef][Medline]

Takizawa Y, Shimizu H, Pulkkinen L, Hiraoka Y, McGrath JA, Suzumori K, Aiso S, et al. (1998b) Novel mutations in the LAMB3 gene shared by two Japanese unrelated families with Herlitz junctional epidermolysis bullosa, and their application for prenatal testing. J Invest Dermatol 110:174–178[CrossRef][Medline]

Takizawa Y, Shimizu H, Pulkkinen L, Nonaka S, Kubo T, Kado Y, Nishikawa T, et al. (1998c) Novel premature termination codon mutations in the laminin {gamma}2-chain gene (LAMC2) in Herlitz junctional epidermolysis bullosa. J Invest Dermatol 111:1233–1234[CrossRef][Medline]

Takizawa Y, Shimizu H, Pulkkinen L, Suzumori K, Kakinuma H, Uitto J, Nishikawa T (1998d) Combination of a novel frameshift mutation (1929delCA) and a recurrent nonsense mutation (W610X) of the LAMB3 gene in a Japanese patient with Herlitz junctional epidermolysis bullosa, and their application for prenatal testing. J Invest Dermatol 111:1239–1241[CrossRef][Medline]

Tiger CF, Champliaud MF, Pedrosa-Domellof F, Thornell LE, Ekblom P, Gullberg D (1997) Presence of laminin {alpha}5 chain and lack of laminin {alpha}1 chain during human muscle development and in muscular dystrophies. J Biol Chem 272:28590–28595[Abstract/Free Full Text]

Uitto J, Pulkkinen L (2001) Molecular genetics of heritable blistering disorders. Arch Dermatol 137:1458–1461[Free Full Text]

Vailly J, Verrando P, Champliaud MF, Gerecke D, Wagman DW, Baudoin C, Aberdam D, et al. (1994) The 100-kDa chain of nicein/kalinin is a laminin B2 chain variant. Eur J Biochem 219: 209–218[Medline]

Yu H, Talts JF (2003) ß1 Integrin and {alpha}-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin {alpha}5 chain G domain. Biochem J 371:289–299[CrossRef][Medline]


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