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• W2008574990 abstract "In the epidermis, β1 integrin expression is normally confined to the basal layer; however, suprabasal expression of β1 integrins in keratinocytes has been found in psoriasis, and it has been suggested that it could be a pathogenic factor of the disease. We have investigated herein the functional state of β1 integrins of human keratinocytes in normal skin and psoriasis. The expression of β1-activation-reporter epitopes was monitored with two monoclonal antibodies, HUTS-21 and MG5A7, that recognize epitopes whose expression parallels functional activity of β1 integrins and correlates with the ligand binding activity of these heterodimeric glycoproteins. We have found that keratinocytes express activation epitopes of β1 integrins, and that these epitopes can be modulated by manganese. The expression of activation epitopes of β1 integrins was related to an enhanced adhesion to fibronectin and collagen. Immunohistochemical studies of normal and psoriatic skin with HUTS-21 and other monoclonal antibodies indicate that, although there is suprabasal expression of β1 integrins in psoriasis, these molecules seem to be in an inactive state. Moreover, most β1 integrins in lateral and apical surfaces of basal keratinocytes of psoriasis are also in a nonactive conformation, implying a decrease of activity compared with normal skin, in which active β1 integrins are distributed all over the basal keratinocytes. In the epidermis, β1 integrin expression is normally confined to the basal layer; however, suprabasal expression of β1 integrins in keratinocytes has been found in psoriasis, and it has been suggested that it could be a pathogenic factor of the disease. We have investigated herein the functional state of β1 integrins of human keratinocytes in normal skin and psoriasis. The expression of β1-activation-reporter epitopes was monitored with two monoclonal antibodies, HUTS-21 and MG5A7, that recognize epitopes whose expression parallels functional activity of β1 integrins and correlates with the ligand binding activity of these heterodimeric glycoproteins. We have found that keratinocytes express activation epitopes of β1 integrins, and that these epitopes can be modulated by manganese. The expression of activation epitopes of β1 integrins was related to an enhanced adhesion to fibronectin and collagen. Immunohistochemical studies of normal and psoriatic skin with HUTS-21 and other monoclonal antibodies indicate that, although there is suprabasal expression of β1 integrins in psoriasis, these molecules seem to be in an inactive state. Moreover, most β1 integrins in lateral and apical surfaces of basal keratinocytes of psoriasis are also in a nonactive conformation, implying a decrease of activity compared with normal skin, in which active β1 integrins are distributed all over the basal keratinocytes. normal human keratinocytes In the epidermis, β1 integrin expression is normally confined to the basal layer; however, there are several situations in which suprabasal, differentiating keratinocytes express β1 integrins. This has been described during wound healing (Hertle et al., 1992Hertle M.D. Kubler M.-D. Leigh I.M. Watt F.M. Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.J Clin Invest. 1992; 89: 1892-1901Crossref PubMed Scopus (207) Google Scholar;Jones et al., 1993Jones J. Sugiyama M. Watt F.M. Speight P.M. Integrin expression in normal, hyperplastic, dysplastic and malignant oral epithelium.J Pathol. 1993; 169: 235-243Crossref PubMed Scopus (120) Google Scholar), and in several cutaneous diseases, including psoriasis (Horrocks et al., 1991Horrocks C. Dunca J.I. Oliver A.M. Thomson A.W. Adhesion molecule expression in psoriatic skin lesions and the influence of cyclosporin A.Clin Exp Immunol. 1991; 84: 157-162Crossref PubMed Scopus (43) Google Scholar;Ralfkiaer et al., 1991Ralfkiaer E. Thomsen K. Vejlsgaard G.L. Expression of a cell adhesion protein (VLA β) in normal and disease skin.Br J Dermatol. 1991; 124: 527-532Crossref PubMed Scopus (18) Google Scholar;Hertle et al., 1992Hertle M.D. Kubler M.-D. Leigh I.M. Watt F.M. Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.J Clin Invest. 1992; 89: 1892-1901Crossref PubMed Scopus (207) Google Scholar;de Pellegrini et al., 1992de Pellegrini G. Luca M. Orecchia G. et al.Expression, topography and function of integrins are severy altered in KC from involved and uninvolved psoriatic skin.J Clin Invest. 1992; 89: 1783-1795Crossref PubMed Scopus (105) Google Scholar;Giannelli et al., 1994Giannelli G. Savoia P. Schivaldi O. de Lospalluti M. Luca M. Marchissio P.C. Quaranta V. Psoriatic lesions in patients with chronic liver disease are distinct from psoriasis vulgaris lesions, as judged on basis of integrin adhesion receptors.Hepatology. 1994; 20: 56-65Crossref PubMed Scopus (17) Google Scholar), eczema, and lichen planus (Ralfkiaer et al., 1991Ralfkiaer E. Thomsen K. Vejlsgaard G.L. Expression of a cell adhesion protein (VLA β) in normal and disease skin.Br J Dermatol. 1991; 124: 527-532Crossref PubMed Scopus (18) Google Scholar). Human keratinocytes express several β1 integrins that include: α2β1, a collagen receptor;α3β1, a laminin and collagen receptor; and α5β1, a receptor for fibronectin (Guo et al., 1991Guo M. Kim L.T. Akiyama S.K. Gralnick H.R. Yamada K.M. Grinnell F. Altered processing of integrin receptors during keratinocyte activation.Exp Cell Res. 1991; 195: 315-322Crossref PubMed Scopus (49) Google Scholar;Watt and Hertle, 1994Watt F.M. Hertle M.D. Keratinocyte integrins.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 153-164Google Scholar). In keratinocytes, β1 integrins not only mediate cell adhesion and migration, but also regulate stratification and the initiation of terminal differentiation (Watt and Hertle, 1994Watt F.M. Hertle M.D. Keratinocyte integrins.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 153-164Google Scholar), and it is a marker of stem cells (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (960) Google Scholar). It has been shown that suprabasal integrin expression is not a direct consequence of inflammation because it is not induced by intradermal injections of cytokines (Hertle et al., 1995Hertle M.D. Jones P.H. Groves R.W. Hudson D.L. Watt F.M. Integrin expression by human epidermal keratinocytes can be modulated by interferon-γ, transforming growth factor-β, tumor necrosis factor-α and culture on a dermal equivalent.J Invest Dermatol. 1995; 104: 260-265Crossref PubMed Scopus (44) Google Scholar), and it has been suggested that suprabasal expression of integrins could be a pathogenic factor in psoriasis (Carroll et al., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Integrin function is regulated at two different levels: selective expression (Guo et al., 1991Guo M. Kim L.T. Akiyama S.K. Gralnick H.R. Yamada K.M. Grinnell F. Altered processing of integrin receptors during keratinocyte activation.Exp Cell Res. 1991; 195: 315-322Crossref PubMed Scopus (49) Google Scholar;Kim et al., 1992Kim L.T. Ishihara S. Lee C.C. Akiyama S.K. Yamada K.M. Grinnell F. Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation.J Cell Sci. 1992; 103: 743-753PubMed Google Scholar) and modulation of its binding properties (Schwartz et al., 1995Schwartz M.A. Schaller M.D. Ginsberg M.H. Integrins: emerging paradigms of signal transduction.Ann Rev Cell Dev Biol. 1995; 11: 549-599Crossref PubMed Scopus (1433) Google Scholar;Sánchez-mateos et al., 1996Sánchez-mateos P. Cabañas C. Sánchez-madrid F. Regulation of integrin function.Sem Cancer Biol. 1996; 7: 99-109Crossref PubMed Scopus (67) Google Scholar). Although most studies have focused on the regulation of expression, recently new antibodies that recognize the activated state of integrins allow the study of the modulation of their binding properties (Lenter et al., 1993Lenter M. Uhlig H. Hamann A. Jenö P. Imhof B. Vestweber D. A monoclonal antibody against an activation epitope on mouse integrin chain β1 blocks adhesion of lymphocytes to the endothelial integrin α6β1.Proc Natl Acad Sci USA. 1993; 90: 9051-9055Crossref PubMed Scopus (219) Google Scholar;Miyake et al., 1994Miyake K. Yamashita Y. Kimoto M. A calcium or manganese-dependent epitope on the integrin β1 chain recognized by a unique MoAb.Int Immunol. 1994; 6: 1221-1226Crossref PubMed Scopus (9) Google Scholar;Bazzoni et al., 1995Bazzoni J. Shih D.-T. Buck C.A. Hemler M.E. Monoclonal antibody 9EG7 defines a novel β1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium.J Biol Chem. 1995; 270: 25570-25577Crossref PubMed Scopus (240) Google Scholar;Mould et al., 1995Mould A.P. Garratt A.N. Askari J.A. Akiyama S.K. Humphries M.J. Identification of a novel anti-integrin monoclonal antibody that recognises a ligand-induced binding site epitope on the β1 subunit.FEBS Lett. 1995; 363: 118-122Abstract Full Text PDF PubMed Scopus (117) Google Scholar;Yednock et al., 1995Yednock T.A. Cannon C. Vandevert C. et al.α4β1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand.J Biol Chem. 1995; 270: 28740-28750Crossref PubMed Scopus (144) Google Scholar). For the β1 family the monoclonal antibody (MoAb) HUTS-21 has been generated and characterized. The epitope recognized by this MoAb is contained within the sequence 355–425 of the β1 chain. Its expression parallels functional activity of VLA integrins and correlates with the ligand binding activity of these heterodimeric glycoproteins (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar). In order to assess the functional state of human keratinocytes in normal and diseased skin, we have studied herein the expression and regulation of β1 activation-reporter epitopes in cultured keratinocytes in normal skin and psoriasis. Normal human keratinocytes (NHK) were cultured, with few modifications, as previously described (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (960) Google Scholar;Parenteau, 1994Parenteau N. Skin equivalents.in: Leigh I.M. Watt F.M. Keratinocyte Methods. Cambridge University Press Cambridge, Cambridge1994: 45-55Google Scholar). In brief, epidermal strips were isolated from the skin of cosmetic surgery using 1.2 U dispase per ml (Boehringer, Mannheim, Germany) for 24 h at 4°C. After incubation of epidermal strips in 0.05% trypsin/0.02% ethylenediamine tetraacetic acid for 25 min at room temperature, keratinocytes were seeded onto plastic dishes at 40,000 cells per cm2. The culture medium consisted of one part Ham's F-12 medium and three parts Dulbecco's modification of Eagle's medium (DMEM, GIBCO, Scotland) supplemented with 10% fetal calf serum, 1.8 × 10–4 M adenine (Sigma, St. Louis, MO), 0.5 μg hydrocortisone per ml (Sigma), 5 μg insulin per ml (Boehringer), 10 ng epidermal growth factor per ml (ICN Biochemicals, Irvine, CA), 5 μg transferrin per ml (Sigma), and 10 ng cholera toxin per ml (ICN Biochemicals). The medium was replaced every other day and cells were passed when almost confluent. Cultures were used between the first and fourth passages. MoAb used in this study directed to the common β1 integrin chain have previously been described: the activating TS2/16 (CD 29) (Hemler et al., 1984Hemler M.E. Sánchez-madrid F. Flotte T.J. et al.Glycoproteins of 210 000 and 130 000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines.J Immunol. 1984; 132: 3011-3018PubMed Google Scholar), the activation-reporter HUTS-21 (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar), and the adhesion-blocking Lia1/2 and VJ1/14 (Campanero et al., 1992Campanero M.R. Arroyo A.G. Pulido R. et al.Functional role of α2/β1 and α4/β1 integrins in leukocyte intercellular adhesion induced through the common β1 subunit.Eur J Immunol. 1992; 22: 3111-3119Crossref PubMed Scopus (63) Google Scholar). MG5A7 is a new anti-β1 MoAb generated by immunizing mice with human umbilical vein endothelial cells, which recognizes an epitope on β1 integrins that appears upon activation of β1 integrins with different stimulatory agents, like Mn2+ cations. The P3X63 mouse myeloma protein (IgG1, kappa) was used as negative control. The HP2/9 anti-CD44 MoAb has been previously described (de la Hera et al., 1989de la Hera A. Acevedo A. Marston W. Sánchez-madrid F. Function of CD44 (Pgp-1) homing receptor in human T cell precursors.Int Immunol. 1989; 1: 598-604Crossref PubMed Scopus (22) Google Scholar). Keratinocytes were detached with 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid, counted, and aliquoted into separate tubes (100,000 cells per tube). Cells were washed in DMEM or HEPES/NaCl buffer (20 mM HEPES, 150 mM NaCl, 2 mg D-glucose per ml, pH 7.4) and resuspended in DMEM or HEPES/NaCl buffer containing the appropriate concentrations of divalent cations or stimuli. The corresponding MoAb was added and cells were incubated for 15 min at 37°C. After this incubation, cells were washed in phosphate buffered saline (PBS) and incubated with 100 μl of 1:100 dilution of fluorescein-isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (DAKO) for 20 min at 4°C. Cells were washed and resuspended in PBS with 10 μg propidium iodide per ml and then analyzed in a Becton Dickinson FACScan flow cytometer. Cell analysis were gated both on forward- and size-scatter intensities and propidium iodide fluorescence to discard dead cells. Keratinocytes were plated in complete medium on glass coverslips and grown until almost confluent. Cells were fixed in 3.7% paraformaldehyde in PBS. Coverslips were then incubated with the primary antibody for 30 min at 37°C, washed, and incubated with the fluorescein-isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (DAKO) at 1:100 for 30 min at 37°C. Specimens were examined with an MRC 1024 Bio-Rad confocal system (Bio-Rad Richmond, CA) mounted on a Zeiss Axiovert 135 microscope (Zeiss, Oberkochen, Germany) equipped with a ×63, 1.4 NA planapochromat objective. Images were stored on magnetoptical disks. Cell adhesion assays were essentially performed as previously described (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar). Briefly, 96 well flat-bottomed plates (Titertek, ICN Biochemicals) were coated with the β1 integrin ligands type 1 collagen (ICN Biochemicals) or fibronectin (Sigma) at the indicated concentrations by overnight incubation at 4°C. After saturation of free plastic sites of wells with 1% boiled bovine serum albumin in PBS for 1 h at room temperature, plates were washed three times with PBS and once with 200 μl of HEPES/NaCl buffer. Fifty microliters of HEPES/NaCl buffer containing 2.5 μg of purified MoAb and/or 2 mM MnCl2 were then added to the wells; 2.5 × 105 keratinocytes in 50 μl of HEPES/NaCl buffer were added to each well and allowed to sediment onto the bottom of the wells for 10 min at 4°C. Plates were then transferred to a CO2 incubator and incubated for 30 min at 37°C. The percentage of cells that remained adhered after three gentle washes of the wells with 200 μl of warm HEPES/NaCl buffer was calculated by measuring the absorbency of the wells at 540 nm after fixation and staining with 0.5% crystal violet in 20% methanol. Eight patients with psoriasis were involved in the study after obtaining an informed consent approved by the Ethical Committee of the Hospital Universitario de la Princesa. Normal skin from excisional surgery from six patients served as controls. A biopsy specimen was obtained from representative areas on extremities under local anesthesia with 2% mepivacaine hydrochloride. All biopsy specimens were snap frozen embedded in OCT (Miles, Naperville, IL) in liquid nitrogen and serially sectioned using a rotary cryostat microtome. Cryostat-cut unfixed sections were mounted on glass slides, covered with acetone for 10 min, dried at room temperature and stored at –70°C. Immunohistochemical staining of psoriatic and normal skin was performed in parallel on frozen sections using a two-step immunoperoxidase technique as described (Fernández-herrera et al., 1989Fernández-herrera J. Sánchez-madrid F. García Díez A. Differential expression of the 4F2 activation antigen on human follicular epithelium in hair cycle.J Invest Dermatol. 1989; 92: 247-250Abstract Full Text PDF PubMed Google Scholar). The sections were incubated in a humidity chamber with the primary monoclonal antibody for 45 min. Slides were rinsed in 1 M Tris-HCl buffer pH 7.6, for 10 min and then covered with rabbit-anti-mouse immunoglobulin conjugated to horseradish peroxidase (DAKO). After two washes in Tris buffer, enzyme staining was developed for 10 min with diaminobenzidine and H2O2. The staining reaction was stopped by washing the slides in Tris buffer. Finally, they were counterstained with hematoxylin for 1 min, rinsed in tap water, dehydrated, and mounted. Sections of skin and keratinocytes in culture were examined by two independent observers. The extent and intensity of staining for each MoAb were recorded and photographs were taken. We explored whether the expression of the activation-dependent conformational epitopes of β1 were regulated in NHK. The expression of β1 was monitored by using TS2/16 and Lia1/2 MoAb, which recognize constitutively expressed epitopes, and HUTS-21 and MG5A7 MoAb, specific for activation-related epitopes. When NHK were incubated at 4°C, the activation β1 epitopes were not detected, whereas a high expression of TS2/16 and Lia1/2 epitopes was observed. Incubation at 37°C showed a significant expression of the activation β1 epitopes, whereas TS2/16 and Lia1/2 expression was not altered. Furthermore, the expression of β1-activation epitopes was also determined on keratinocytes under conditions of divalent cations described to change β1 integrins to a high affinity state. Mn2+ has been described as one of the most important activating cations of β1 integrins (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar). We found an enhanced expression of the activation epitopes detected by HUTS-21 and MG5A7 (Figure 1a). The induced expression of the activation epitopes correlated with the concentration of Mn2+ used (Figure 1b). Although Mn2+ has been described to enhance adhesion of T lymphoblasts to fibronectin and type I collagen (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar), we performed adhesion studies to explore if the increased expression of HUTS-21 induced by Mn2+ was correlated by changes in the adhesion of keratinocytes through β1 integrins (Figure 2). We found that both TS2/16 and Mn2+ greatly enhance the adhesion of NHK to fibronectin (0.5–1 μg per ml) and type I collagen (0.25–0.5 μg per ml). We also found that the MoAb VJ1/14, a very potent inhibitory anti-β1 MoAb, almost reversed the enhanced adhesion induced by Mn2+. Immunofluorescence digital confocal microscopy studies of cultured NHK showed different patterns of expression with the anti-β1 antibodies studied. Both TS2/16 and Lia1/2 showed fluorescence in all four optical sections taken at a pitch of 0.5 μm indicating expression all over the cell membrane, with an enhancement of the fluorescence in the intercellular surfaces (Figure 3a–d). In contrast, activated epitopes on β1 integrins assessed by HUTS-21 and MG5A7 MoAb showed little intercellular staining. Conspicuous linear arrangement of fluorescence in the basal surface of the cell was found in most keratinocytes with both antibodies (Figure 3e, f). No staining was found in other optical sections of the cell above the basal surface. The negative control P3X63 did not show any relevant staining. We found that in normal skin, β1 integrins, evaluated by TS2/16 and Lia1/2, were present only in the basal cell layer of keratinocytes, and these cells were stained throughout the cell with conspicuous staining in the membrane (Figure 4a, b). Dermal endothelium showed a strong reactivity similar to the basal cell layer. HUTS 21 and MG5A7 antibodies stained only the basal cell layer of normal skin, and keratinocytes were stained all over the cell, both at the membrane and at the cytoplasm (Figures 4c, d, 5a, b). Capillaries and other endothelia showed similar staining to that found on the epidermis. In psoriatic skin, there was a strong expression of β1 integrins in the basal cell layer, predominantly on the membrane. A consistent staining could be found in keratinocytes of suprabasal layers with TS2/16 and Lia1/2 antibodies but only in the tips of dermal papillae (Figure 4e, f). The endothelium of capillary vessels showed a similar staining to that found in the basal cell layer. Activation β1 epitopes were detected consistently only in the dermo–epidermal junction, with scant or no staining in the rest of the cell (Figures 4g, h, 5d, e). Endothelium showed a stronger staining, which was similar to what we found in normal skin. As a control for staining intensity we included the CD44 hyaluronate receptor (Figure 5c, f). A comparable CD44 staining pattern was found in both normal and psoriatic skin. Integrins are important molecules in cell–cell and cell–extracellular matrix interactions and regulate cell functions and behavior in many cell types (Schwartz et al., 1995Schwartz M.A. Schaller M.D. Ginsberg M.H. Integrins: emerging paradigms of signal transduction.Ann Rev Cell Dev Biol. 1995; 11: 549-599Crossref PubMed Scopus (1433) Google Scholar). Cellular adhesive properties are regulated through the selective expression of integrins (Guo et al., 1991Guo M. Kim L.T. Akiyama S.K. Gralnick H.R. Yamada K.M. Grinnell F. Altered processing of integrin receptors during keratinocyte activation.Exp Cell Res. 1991; 195: 315-322Crossref PubMed Scopus (49) Google Scholar) as well as by the modulation of the binding properties of these receptors. The avidity of integrins for their ligands is not constant and is dynamically regulated (Sánchez-mateos et al., 1996Sánchez-mateos P. Cabañas C. Sánchez-madrid F. Regulation of integrin function.Sem Cancer Biol. 1996; 7: 99-109Crossref PubMed Scopus (67) Google Scholar). The existence of different conformations of β1 integrins in vivo has been inferred from both cell adhesion studies with keratinocytes and the observations recently described on the expression of activated β1 integrins in vitro (Kim and Yamada, 1997Kim L.T. Yamada K.M. Evidence that β1 integrins in keratinocyte cell-cell junctions are not in the ligand-occupied conformation.J Invest Dermatol. 1997; 108: 876-880Abstract Full Text PDF PubMed Scopus (17) Google Scholar); however, a direct assessment of the expression of activated β1 in normal and diseased skin has not been explored so far. The use of the HUTS-21 and MG5A7 MoAb, which detect activation-dependent epitopes of β1 integrins (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar), has facilitated the study of activated β1 expression both in vivo and in vitro. These MoAb recognize epitopes on a regulatory region of the common β1 subunit of VLA integrins whose expression parallels functional activation and ligand binding by these adhesion receptors (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar;Gómez et al., 1997Gómez M. Luque A. del Pozo M.A. Hogg N. Sánchez-madrid F. Cabañas C. Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins.Eur J Immunol. 1997; 27: 8-16Crossref PubMed Scopus (34) Google Scholar). We have found that keratinocytes express activation epitopes of β1 integrins, we have shown that these epitopes can be modulated by manganese, an inducer of adhesion of keratinocytes (Hotchin et al., 1993Hotchin N.A. Kovach N.L. Watt F.M. Functional down-regulation of α5β1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not.J Cell Sci. 1993; 106: 1131-1138PubMed Google Scholar) and a potent inducer of activation epitopes in β1 integrins (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar), and we have shown that these epitopes are related to adhesion to fibronectin and type I collagen. These data are in agreement with previous reported observations with T lymphocytes (Luque et al., 1996Luque A. Gómez M. Puzon W. Takada Y. Sánchez-madrid F. Cabañas C. Activated conformations of VLA integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355–425) of the common β1 chain.J Biol Chem. 1996; 271: 11067-11075Crossref PubMed Scopus (255) Google Scholar). In cultured NHK, our results showed that, whereas β1 integrins can be found all over the keratinocyte membrane, activation epitopes appear mainly in a linear arrangement on the basal surface of cells where focal adhesions are formed (Kim and Yamada, 1997Kim L.T. Yamada K.M. Evidence that β1 integrins in keratinocyte cell-cell junctions are not in the ligand-occupied conformation.J Invest Dermatol. 1997; 108: 876-880Abstract Full Text PDF PubMed Scopus (17) Google Scholar). This distribution could be the reflection of the interactions with extracellular matrix ligands, similar to what has been described in human lymphocytes (Sánchez-mateos et al., 1996Sánchez-mateos P. Cabañas C. Sánchez-madrid F. Regulation of integrin function.Sem Cancer Biol. 1996; 7: 99-109Crossref PubMed Scopus (67) Google Scholar;Gómez et al., 1997Gómez M. Luque A. del Pozo M.A. Hogg N. Sánchez-madrid F. Cabañas C. Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins.Eur J Immunol. 1997; 27: 8-16Crossref PubMed Scopus (34) Google Scholar). Under physiologic conditions, HUTS-21 detects a ligand induced binding site epitope on β1 (Gómez et al., 1997Gómez M. Luque A. del Pozo M.A. Hogg N. Sánchez-madrid F. Cabañas C. Functional relevance during lymphocyte migration and cellular localization of activated β1 integrins.Eur J Immunol. 1997; 27: 8-16Crossref PubMed Scopus (34) Google Scholar). In addition, stimulating agents acting from without the cell, such as Mn2+ and activating TS2/16 MoAb, can impose a similar conformation to that induced by ligand occupancy. Expression of β1 integrin has been related to the proliferative state of keratinocytes (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (960) Google Scholar;Bata-csorgo et al., 1995Bata-csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (180) Google Scholar). Parallel to accelerated proliferation, the β1 integrin expression became very bright in vitro. As the proliferation slowed down on confluent cultures, the intensity diminished (Bata-csorgo et al., 1995Bata-csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (180) Google Scholar). Moreover, β1 integrins are downregulated when keratinocytes initiate terminal differentiation (Jones and Watt, 1993Jones P.H. Watt F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (960) Google Scholar;Hodivala and Watt, 1994Hodivala K.J. Watt F.M. Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation.J Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (201) Google Scholar). Nevertheless, keratinocytes in vitro have a high proliferative rate (Rheinwald and Green, 1977Rheinwald J.G. Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.Nature. 1977; 265: 421-424Crossref PubMed Scopus (806) Google Scholar) similar to psoriatic epidermis, and express β1 integrins together with several markers of differentiation (peanut agglutinin, involucrin, K1/K10 keratins) (Hodivala and Watt, 1994Hodivala K.J. Watt F.M. Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation.J Cell Biol. 1994; 124: 589-600Crossref PubMed Scopus (201) Google Scholar;Bata-csorgo et al., 1995Bata-csorgo Z. Hammerberg C. Voorhees J.J. Cooper K.D. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture.J Clin Invest. 1995; 95: 317-327Crossref PubMed Scopus (180) Google Scholar). Transgenic mice with suprabasal expression of integrins have shown features reminiscent of chronic plaque psoriasis, suggesting that the relationship between hyperproliferation and suprabasal β1 integrin expression could be causal (Carroll et al., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Nevertheless, the functional state of these β1 integrins has not been formally addressed. Our immunohistochemical studies with MoAb that recognize activated β1 integrins indicate that, although there is suprabasal expression of β1 integrins in psoriasis, these molecules seem to be in an inactive state. Moreover, most β1 integrins in lateral and apical surfaces of basal keratinocytes are also in a nonactive conformation, implying a decrease of activity compared with normal skin, in which active β1 integrins are distributed all over the basal keratinocytes. Our results with cultured NHK, showing intense β1 fluorescence on cell–cell contacts and the apical surface of keratinocytes but unrelated to the expression of HUTS-21 or MG5A7, suggest a similar pattern of expression to that found in psoriatic skin. Probably, the psoriatic-like behavior of NHK in culture could explain the discrepancy in the staining patterns. It has been proposed that the presence of integrins in the differentiating cell layers stimulates proliferation in the basal layer, perhaps by signaling that there is a deficit in the size of the differentiated compartment that should be replenished through increased proliferation (Carroll et al., 1995Carroll J.M. Romero M.R. Watt F.M. Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (275) Google Scholar). Our results show that the expression of β1 is not correlated with its functional state, thus indicating that downregulation of the β1 functional activity occurs in psoriasis. Suprabasal expression of inactive β1 might be related to the hyperproliferative state of keratinocytes. β1 integrins mediate keratinocyte adhesion to extracellular matrix components (Watt and Hertle, 1994Watt F.M. Hertle M.D. Keratinocyte integrins.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 153-164Google Scholar), and its downregulation may be one of the first steps in the differentiation process that leads keratinocytes to the spinous layer (Adams and Watt, 1990Adams J.C. Watt F.M. Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes α5β1 integrin loss from the cell surface.Cell. 1990; 63: 425-435Abstract Full Text PDF PubMed Scopus (390) Google Scholar). The mechanisms leading to the elimination of β1 from the membrane (Dalton et al., 1995Dalton S.L. Scharf E. Briesewitz R. Marcantonio E.E. Assoian R.K. Cell adhesion to extracellular matrix regulates the life cycle of integrins.Mol Biol Cell. 1995; 6: 1781-1791Crossref PubMed Scopus (70) Google Scholar;Hotchin et al., 1995Hotchin N.A. Gandarillas A. Watt F.M. Regulation of cell surface β1 integrin levels during keratinocyte terminal differentiation.J Cell Biol. 1995; 128: 1209-1219Crossref PubMed Scopus (123) Google Scholar) could not be so efficient to destroy the protein in the short time it takes keratinocytes to migrate from the basal to the suprabasal layers. Expression of β1 integrins in lateral and apical surfaces of NHK in culture and in skin biopsies have suggested its implication in cell–cell interactions (Carter et al., 1990Carter W.G. Wayner E.A. Bouchard T.S. Kaur P. The role of integrins α2β1 and α3β1 in cell-cell and cell-substrate adhesion of human epidermal cells.J Cell Biol. 1990; 110: 1387-1404Crossref PubMed Scopus (505) Google Scholar;Larjava et al., 1990Larjava H. Peltonen J. Akiyama S.K. Yamada S.S. Gralnick H.R. Uitto J. Yamada K.M. Novel function for β1 integrins in keratinocyte cell–cell interactions.J Cell Biol. 1990; 110: 803-815Crossref PubMed Scopus (248) Google Scholar;Watt and Hertle, 1994Watt F.M. Hertle M.D. Keratinocyte integrins.in: Leigh I.M. Lane E.B. Watt F.M. The Keratinocyte Handbook. Cambridge University Press, Cambridge1994: 153-164Google Scholar). Although studies in cultured NHK with other anti-β1 activation reporter MoAb (Kim and Yamada, 1997Kim L.T. Yamada K.M. Evidence that β1 integrins in keratinocyte cell-cell junctions are not in the ligand-occupied conformation.J Invest Dermatol. 1997; 108: 876-880Abstract Full Text PDF PubMed Scopus (17) Google Scholar) has shown that β1 integrins in cell–cell contact are not in the ligand-occupied conformation, as we found with HUTS 21 and MG5A7, our results in normal skin imply that these integrins are, at least partially, in an activated state. A decrease of activation epitopes expression in psoriasis, a disease of keratinocyte hyperproliferation, suggests that activated β1 integrins on lateral and apical surfaces of the cell might be signalling inhibition of proliferation. This work was supported by grants from the Fondo de Investigación Sanitaria 95/0196 and 96/2009 and by a fellowship from the Fondo de Investigación Sanitaria (to G. F. B.)" @default.
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