Alpha 6 integrins are required for Langerhans cell migration from the epidermis - PubMed (original) (raw)

Alpha 6 integrins are required for Langerhans cell migration from the epidermis

A A Price et al. J Exp Med. 1997.

Abstract

Topical exposure of mice to chemical allergens results in the migration of epidermal Langerhans cells (LCs) from the skin and their accumulation as immunostimulatory dendritic cells (DCs) in draining lymph nodes. Epidermal cell-derived cytokines have been implicated in the maturation and migration of LCs, but the adhesion molecules that regulate LC migration have not been studied. We hypothesized that integrin-mediated interactions with extracellular matrix components of the skin and lymph node may regulate LC/DC migration. We found that alpha 6 integrins and alpha 4 integrins were differentially expressed by epidermal LCs and lymph node DCs. A majority of LCs (70%) expressed the alpha 6 integrin subunit, whereas DCs did not express alpha 6 integrins. In contrast, the alpha 4 integrin subunit was expressed at high levels on DCs but at much lower levels on LCs. The anti-alpha 6 integrin antibody, GoH3, which blocks binding to laminin, completely prevented the spontaneous migration of LCs from skin explants in vitro and the rapid migration of LCs from mouse ear skin induced after intradermal administration of TNF-alpha in vivo. GoH3 also reduced the accumulation of DCs in draining lymph nodes by a maximum of 70% after topical administration of the chemical allergen oxazolone. LCs remaining in the epidermis in the presence of GoH3 adopted a rounded morphology, rather than the interdigitating appearance typical of LCs in naive skin, suggesting that the cells had detached from neighboring keratinocytes and withdrawn cellular processes in preparation for migration, but were unable to leave the epidermis. The anti-alpha 4 integrin antibody PS/2, which blocks binding to fibronectin, had no effect on LC migration from the epidermis either in vitro or in vivo, or on the accumulation of DCs in draining lymph nodes after oxazolone application. RGD-containing peptides were also without effect on LC migration from skin explants. These results identify an important role for alpha 6 integrins in the migration of LC from the epidermis to the draining lymph node by regulating access across the epidermal basement membrane. In contrast, alpha 4 integrins, or other integrin-dependent interactions with fibronectin that are mediated by the RGD recognition sequence, did not influence LC migration from the epidermis. In addition, alpha 4 integrins did not affect the accumulation of LCs as DCs in draining lymph nodes.

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Figures

Figure 1

Figure 1

(A) Expression of α6 and β4 integrin subunits on LCs. Epidermal cell suspensions were prepared from the ears of naive mice by treatment with 0.5% trypsin and LCs were identified by the expression of MHC class II. The expression of integrin subunits was determined using antibodies to α6 (GoH3 or EA-1) or β4 subunits in comparison with an isotype-matched control antibody (IgG2a). Results show log fluorescence (0–104 channels) for MHC class II (x axis) and integrin subunit (y axis). The percentage of cells within each quadrant is given. (B) Expression of α4, α6, and β4 integrin subunits on lymph node DCs. DCs were enriched from the draining lymph nodes of oxazolone-treated mice by density gradient centrifugation on Metrizamide. DCs were distinguished from lymphocytes by forward scatter (FSC) versus side scatter (SSC) analysis and identified by high expression of MHC class II and dual staining for NLDC145 antigen. Histograms show the expression of α4, α6, and β4 integrin subunits on MHC class II positive DCs (solid lines) in comparison with isotype-matched control antibodies (dashed lines). The profiles show log fluorescence (0–104 channels) on the x axis and cell number (0–40) on the y axis.

Figure 1

Figure 1

(A) Expression of α6 and β4 integrin subunits on LCs. Epidermal cell suspensions were prepared from the ears of naive mice by treatment with 0.5% trypsin and LCs were identified by the expression of MHC class II. The expression of integrin subunits was determined using antibodies to α6 (GoH3 or EA-1) or β4 subunits in comparison with an isotype-matched control antibody (IgG2a). Results show log fluorescence (0–104 channels) for MHC class II (x axis) and integrin subunit (y axis). The percentage of cells within each quadrant is given. (B) Expression of α4, α6, and β4 integrin subunits on lymph node DCs. DCs were enriched from the draining lymph nodes of oxazolone-treated mice by density gradient centrifugation on Metrizamide. DCs were distinguished from lymphocytes by forward scatter (FSC) versus side scatter (SSC) analysis and identified by high expression of MHC class II and dual staining for NLDC145 antigen. Histograms show the expression of α4, α6, and β4 integrin subunits on MHC class II positive DCs (solid lines) in comparison with isotype-matched control antibodies (dashed lines). The profiles show log fluorescence (0–104 channels) on the x axis and cell number (0–40) on the y axis.

Figure 2

Figure 2

The effect of anti-α6 and anti-α4 integrin antibodies on the migration of epidermal LCs in vitro. Skin explants were derived from the ears of naive mice and incubated on medium containing either (A) anti-α6 (GoH3) or (B) anti-α4 (PS/2) integrin antibodies at 10, 50, or 100 μg/ml (solid bars). Controls included explants cultured on medium containing either no antibody (open bars) or isotype-matched control antibody at 100 μg/ml (hatched bars). Epidermal sheets were prepared from fresh ear skin (0 h) and explants after 72 h of incubation and the number of LCs/mm2 determined by immunohistochemistry. The results are expressed as means ± SD (n = 18). At all concentrations tested, in cultures containing anti-α6 antibodies, the frequency of LCs did not differ significantly from that found in fresh explants. These same values were significantly (P <0.0001) higher than those found in fresh explants cultured for 72 h in the absence of antibody, or with an isotype-matched control antibody (A). Similar treatment of explant cultures with anti-α4 antibody failed to result, at any concentration, in a significant increase in LC numbers compared with controls (B).

Figure 3

Figure 3

Immunohistochemical staining of epidermal LCs in situ. Epidermal sheets were prepared either from (A) naive mice, or from skin explants that had been incubated for 72 h on (B) culture medium containing anti-α6 integrin antibody GoH3 at 50 μg/ml, or (C) culture medium alone. LC are stained for MHC class II using indirect immunoperoxidase staining. Note rounded morphology of LCs in anti-α6 integrin antibody– treated explants in comparison with interdigitating morphology of LCs in naive skin. Magnification, ×600.

Figure 3

Figure 3

Immunohistochemical staining of epidermal LCs in situ. Epidermal sheets were prepared either from (A) naive mice, or from skin explants that had been incubated for 72 h on (B) culture medium containing anti-α6 integrin antibody GoH3 at 50 μg/ml, or (C) culture medium alone. LC are stained for MHC class II using indirect immunoperoxidase staining. Note rounded morphology of LCs in anti-α6 integrin antibody– treated explants in comparison with interdigitating morphology of LCs in naive skin. Magnification, ×600.

Figure 3

Figure 3

Immunohistochemical staining of epidermal LCs in situ. Epidermal sheets were prepared either from (A) naive mice, or from skin explants that had been incubated for 72 h on (B) culture medium containing anti-α6 integrin antibody GoH3 at 50 μg/ml, or (C) culture medium alone. LC are stained for MHC class II using indirect immunoperoxidase staining. Note rounded morphology of LCs in anti-α6 integrin antibody– treated explants in comparison with interdigitating morphology of LCs in naive skin. Magnification, ×600.

Figure 4

Figure 4

The effect of anti-α6 integrin antibody EA-1 and GRGDS peptide on the migration of epidermal LCs in vitro. Skin explants were incubated on culture medium containing: (A) 50 μg/ml EA-1 (solid bars) or control antibody (hatched bars); (B) 500 μM GRGDS (RGD; solid bars) or GRDGS (RDG; hatched bars). Additional controls included explants incubated on medium containing no antibody (open bars). The number of LCs/ mm2 was determined after 24 and 72 h of incubation and compared with fresh skin (0 h). Results are expressed as means ± SD (n = 18). Treatment of explant cultures with EA-1 antibody failed to cause a significant increase in the frequency of LCs at 24 or 72 h compared with cultures containing no antibody or an isotype-matched control antibody (A). Addition to explant cultures of GRGDS also failed to cause a significant increase in the frequency of LCs at 24 or 72 h compared with cultures containing no peptide or GRDGS.

Figure 5

Figure 5

The effect of anti-α6 and anti-α4 integrin antibodies on TNF-α–induced epidermal LC migration in vivo. Groups of four mice received 40 μg of (A) anti-α6 integrin (GoH3) or (B) anti-α4 integrin (PS/2) antibody intraperitoneally. Controls either received isotype-matched control antibody or were left untreated. 2 h after administration of antibody, two mice per group received 30 μl injection intradermally into both ear pinnae of 50 ng murine TNF-α in 0.1% BSA. The remaining two mice received 30 μl intradermal injection of 0.1% BSA. Ears were removed 30 min later, epidermal sheets were prepared, and the number of LCs/mm2 was determined by immunofluorescence analysis. Results are means ± SD (n = 40). Treatment of mice with TNF-α (in both A and B) caused a significant decrease in the frequency of LCs compared with either untreated controls or mice exposed to BSA alone (P <0.005). Treatment with anti-α6 resulted in a significantly higher frequency of LC compared with mice exposed to TNF-α together with an isotype-matched control antibody (P <0.005; A). In contrast, anti-α4 failed to affect significantly LC frequency compared with isotype controls.

Figure 6

Figure 6

Immunofluorescence staining of epidermal LCs in situ. Groups of mice (n = 2) received a single 100 μl injection intraperitoneally of 40 μg anti-α6 integrin antibody (GoH3) 2 h before intradermal injection into both ear pinnae of (A) 50 ng murine recombinant TNF-α or (B) 0.1% BSA alone. Ears were removed 30 min later, epidermal sheets were prepared, and the morphology of LCs was assessed after indirect immunofluorescence staining for MHC class II expression. Note the rounded morphology of LCs in TNF-α–treated ears in comparison with interdigitating morphology of LCs in controls. Magnification, ×800.

Figure 6

Figure 6

Immunofluorescence staining of epidermal LCs in situ. Groups of mice (n = 2) received a single 100 μl injection intraperitoneally of 40 μg anti-α6 integrin antibody (GoH3) 2 h before intradermal injection into both ear pinnae of (A) 50 ng murine recombinant TNF-α or (B) 0.1% BSA alone. Ears were removed 30 min later, epidermal sheets were prepared, and the morphology of LCs was assessed after indirect immunofluorescence staining for MHC class II expression. Note the rounded morphology of LCs in TNF-α–treated ears in comparison with interdigitating morphology of LCs in controls. Magnification, ×800.

Figure 7

Figure 7

The effect of (A) anti-α6 and (B) anti-α4 integrin antibodies on oxazolone-induced DC accumulation in lymph nodes in vivo. Antibody was administered to groups of 10 mice in single 100 μl injections intraperitoneally for the 10, 20, and 200 μg doses or in single 30 μl injections intradermally into the dorsum of both ears for 12 μg dose. The 200 μg dose of anti-α4 integrin antibody was given in 2 × 100 μg doses 2 h before and 8 h after oxazolone treatment. Each graph represents a separate experiment and the amount of antibody received per mouse, and the method of administration is indicated below each graph. 2 h after antibody administration, mice received 25 μl of 0.5% oxazolone on the dorsum of both ears. Draining auricular lymph nodes were excised 18 h later and the number of DCs per lymph node was determined. Results for isotype-matched control antibody (hatched bars) and integrin antibody (solid bars) are compared with untreated, naive mice (open bars).

Figure 7

Figure 7

The effect of (A) anti-α6 and (B) anti-α4 integrin antibodies on oxazolone-induced DC accumulation in lymph nodes in vivo. Antibody was administered to groups of 10 mice in single 100 μl injections intraperitoneally for the 10, 20, and 200 μg doses or in single 30 μl injections intradermally into the dorsum of both ears for 12 μg dose. The 200 μg dose of anti-α4 integrin antibody was given in 2 × 100 μg doses 2 h before and 8 h after oxazolone treatment. Each graph represents a separate experiment and the amount of antibody received per mouse, and the method of administration is indicated below each graph. 2 h after antibody administration, mice received 25 μl of 0.5% oxazolone on the dorsum of both ears. Draining auricular lymph nodes were excised 18 h later and the number of DCs per lymph node was determined. Results for isotype-matched control antibody (hatched bars) and integrin antibody (solid bars) are compared with untreated, naive mice (open bars).

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