Amphiregulin and Epidermal Hyperplasia
2005; Elsevier BV; Volume: 166; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)62322-x
ISSN1525-2191
AutoresNarasimharao Bhagavathula, Kamalakar C. Nerusu, Gary J. Fisher, Gao Liu, Archana Thakur, L Gemmell, S. Madan Kumar, Zenghai H. Xu, Paul R. Hinton, Naoya Tsurushita, Nicholas F. Landolfi, John J. Voorhees, James Varani,
Tópico(s)T-cell and B-cell Immunology
ResumoOverexpression of amphiregulin has been shown to induce psoriasiform changes in the skin of transgenic mice shortly after birth. Therefore, amphiregulin has been suggested as a target for anti-psoriatic therapy. To test this theory, a humanized monoclonal antibody capable of neutralizing human amphiregulin was examined for anti-proliferative effects in the human skin-severe combined immunodeficient (SCID) mouse transplant model. The anti-amphiregulin antibody reduced epidermal thickness of transplanted psoriatic skin and also inhibited the hyperplastic response that developed in nonpsoriatic skin after transplantation. The same antibody also suppressed keratinocyte proliferation in monolayer culture in a dose-dependent manner. Under the same conditions in which keratinocyte proliferation was inhibited, the antibody had little effect on proliferation of human dermal fibroblasts and no effect on type I procollagen production by these cells. Taken together, these data indicate an important role for amphiregulin in psoriatic hyperplasia and suggest that inhibition of amphiregulin activity could be an efficacious therapeutic strategy for psoriasis. These data also suggest that the hyperplastic response occurring in nonpsoriatic human skin on transplantation to the SCID mouse is mediated, in large part, by amphiregulin. Overexpression of amphiregulin has been shown to induce psoriasiform changes in the skin of transgenic mice shortly after birth. Therefore, amphiregulin has been suggested as a target for anti-psoriatic therapy. To test this theory, a humanized monoclonal antibody capable of neutralizing human amphiregulin was examined for anti-proliferative effects in the human skin-severe combined immunodeficient (SCID) mouse transplant model. The anti-amphiregulin antibody reduced epidermal thickness of transplanted psoriatic skin and also inhibited the hyperplastic response that developed in nonpsoriatic skin after transplantation. The same antibody also suppressed keratinocyte proliferation in monolayer culture in a dose-dependent manner. Under the same conditions in which keratinocyte proliferation was inhibited, the antibody had little effect on proliferation of human dermal fibroblasts and no effect on type I procollagen production by these cells. Taken together, these data indicate an important role for amphiregulin in psoriatic hyperplasia and suggest that inhibition of amphiregulin activity could be an efficacious therapeutic strategy for psoriasis. These data also suggest that the hyperplastic response occurring in nonpsoriatic human skin on transplantation to the SCID mouse is mediated, in large part, by amphiregulin. Psoriasis is an inflammatory skin disease, affecting 2 to 3% of the population.1Krueger GC Bergstresser PR Lowe NJ Voorhees JJ Weinstein GD Psoriasis.J Am Acad Dermatol. 1984; 11: 937-947Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 2Fry L Psoriasis.Br J Dermatol. 1988; 119: 445-461Crossref PubMed Scopus (157) Google Scholar, 3Sander HM Morris LF Phillips CM Harrison PE Menter A The annual cost of psoriasis.J Am Acad Dermatol. 1993; 128: 422-425Abstract Full Text PDF Scopus (125) Google Scholar, 4Weiss SC Kimball AB Liewehr DJ Blauvelt A Turner ML Emanuel EJ Quantifying the harmful effect of psoriasis on health-related quality of life.J Am Acad Dermatol. 2002; 47: 512-518Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar It is characterized by excessive keratinocyte proliferation, leading to a significant thickening of the epidermis, expansion of epidermal rete pegs into papillary dermal space, abnormalities in the differentiation process, and continuous shedding of the thickened epidermis. The etiology of the disease is complex and not well understood. T cells are almost certainly involved in the initiation and maintenance of psoriatic lesions.5Valdimarsson H Baker BS Jonsdottir I Powles A Fry L Psoriasis: a T-cell mediated autoimmune disease mediated by streptococcal superantigen?.Immunol Today. 1995; 16: 145-149Abstract Full Text PDF PubMed Scopus (339) Google Scholar, 6Austin LM Ozawa M Kikuchi T Walters IB Kueuger JG The majority of epidermal T cells in psoriasis vulgaris lesions can produce type 1 cytokines—interferon-γ, interleukin-2 and tumor necrosis factor-α—defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias also measured in circulating blood T cells in psoriasis patients.J Invest Dermatol. 1999; 113: 101-108Crossref Scopus (428) Google Scholar, 7Baker BS Brent L Valdimarsson H Powles AV al-Imara L Walker M Fry L Is epidermal proliferation in psoriatic skin grafts on nude mice driven by T-cell derived cytokines?.Br J Dermatol. 1992; 126: 105-110Crossref PubMed Scopus (53) Google Scholar, 8Wong RL Winslow CM Cooper KD The mechanisms of action of cyclosporin A in the treatment of psoriasis.Immunol Today. 1993; 14: 69-74Abstract Full Text PDF PubMed Scopus (126) Google Scholar Activated T cells in the region of the dermal-epidermal junction are thought to drive the hyperplastic proliferative response through elaboration of TH1 cytokines including tumor necrosis factor-α, interferon-γ, interleukin-6, and interleukin-8.6Austin LM Ozawa M Kikuchi T Walters IB Kueuger JG The majority of epidermal T cells in psoriasis vulgaris lesions can produce type 1 cytokines—interferon-γ, interleukin-2 and tumor necrosis factor-α—defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias also measured in circulating blood T cells in psoriasis patients.J Invest Dermatol. 1999; 113: 101-108Crossref Scopus (428) Google Scholar, 7Baker BS Brent L Valdimarsson H Powles AV al-Imara L Walker M Fry L Is epidermal proliferation in psoriatic skin grafts on nude mice driven by T-cell derived cytokines?.Br J Dermatol. 1992; 126: 105-110Crossref PubMed Scopus (53) Google Scholar, 9Dustin ML Singer KH Tuck DT Springer TA Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon-gamma and is mediated by intercellular adhesion molecule-1.J Exp Med. 1988; 167: 1323-1340Crossref PubMed Scopus (577) Google Scholar The prominent role of immune cells in psoriatic pathology has biased current therapeutic development efforts toward manipulating the immune system, resulting in the undesirable consequences of immunosuppression. Abnormalities in keratinocyte function also appear to be important to the overall pathophysiology of the disease. Keratinocytes from psoriatic lesional skin have been shown to be less responsive to the growth-inhibitory effects of interferon-γ than normal keratinocytes10Nickoloff BJ Mitra RS Elder JT Fisher GJ Voorhees JJ Decreased growth inhibition by recombinant gamma interferon is associated with increased production of transforming growth factor alpha in keratinocytes cultured from psoriatic lesions.Br J Dermatol. 1989; 121: 161-174Crossref PubMed Scopus (103) Google Scholar and differences in cytokine generation between normal and psoriatic keratinocytes have been documented.10Nickoloff BJ Mitra RS Elder JT Fisher GJ Voorhees JJ Decreased growth inhibition by recombinant gamma interferon is associated with increased production of transforming growth factor alpha in keratinocytes cultured from psoriatic lesions.Br J Dermatol. 1989; 121: 161-174Crossref PubMed Scopus (103) Google Scholar, 11Nickoloff BJ The cytokine network in psoriasis.Arch Dermatol. 1991; 127: 871-884Crossref PubMed Scopus (317) Google Scholar, 12Goebeler M Toksoy A Spandau U Engelhardt E Broker EB Gillitzer R The C-X-C chemokine Mig is highly expressed in the papillae of psoriatic lesions.J Pathol. 1998; 184: 89-95Crossref PubMed Scopus (77) Google Scholar A prominent role for epidermal growth factor (EGF) receptor function in psoriatic hyperplasia is strongly suggested. Past studies13Varani J Kang S Stoll S Elder JT Human psoriatic skin in organ culture: comparison with normal skin exposed to exogenous growth factors and effects of an antibody to the EGF receptor.Pathobiology. 1998; 66: 253-259Crossref PubMed Scopus (38) Google Scholar demonstrated that treatment of psoriatic lesional skin in organ culture with an antibody directed against the EGF receptor ameliorated abnormal histological features. Conversely, when skin from nonpsoriatic individuals or nonlesional skin from individuals with psoriasis was maintained in organ culture and treated with EGF, histological features similar to those of psoriatic lesional skin develop. Additionally, several ligands for the EGF receptor, including transforming growth factor-α, heparin-binding EGF and amphiregulin, are elevated in psoriatic lesional skin relative to control skin.14Gottlieb AB Chang CK Posnett DN Fanelli B Tam JP Detection of transforming growth factor-alpha in normal, malignant and hyperproliferative human keratinocytes.J Exp Med. 1988; 167: 670-675Crossref PubMed Scopus (198) Google Scholar, 15Elder JT Fisher GJ Lindquist PB Bennett GJ Pittelkow MR Coffey RJ Ellingsworth L Derynck R Voorhees JJ Overexpression of transforming growth factor-α in psoriatic epidermis.Science. 1989; 243: 811-814Crossref PubMed Scopus (501) Google Scholar, 16Piepkorn M Pittelkow MR Cook PW Autocrine regulation of keratinocytes: the emerging role of heparin-binding, epidermal growth factor-related growth factors.J Invest Dermatol. 1998; 111: 715-721Crossref PubMed Scopus (121) Google Scholar, 17Piepkorn M Predd H Underwood R Cook P Proliferation-differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes.Arch Dermatol Res. 2003; 295: 93-101Crossref PubMed Scopus (63) Google Scholar, 18Cook PW Pittelkow MR Keeble WW Graves-Deal R Coffey Jr, RJ Shipley GD Amphiregulin messenger RNA is elevated in psoriatic epidermis and gastrointestinal carcinomas.Cancer Res. 1992; 52: 3224-3227PubMed Google Scholar Amphiregulin may be the key EGF receptor ligand in psoriasis. Studies by Cook and colleagues19Cook PW Piepkorn M Clegg CH Plowman GD DeMay JM Brown JR Pittelkow MR Transgenic expression of the human amphiregulin gene induces a psoriasis-like phenotype.J Clin Invest. 1997; 100: 2286-2294Crossref PubMed Scopus (195) Google Scholar, 20Cook PW Brown JR Cornell KA Pittelkow MR Suprabasal expression of human amphiregulin in the epidermis of transgenic mice induces a severe, early-onset, psoriasis-like skin pathology: expression of amphiregulin in the basal epidermis is also associated with synovitis.Exp Dermatol. 2004; 13: 347-356Crossref PubMed Scopus (74) Google Scholar have demonstrated that in two amphiregulin-overexpressing transgenic mouse models, psoriasiform changes are observed in the skin shortly after birth. In one of the models, targeting amphiregulin overexpression to the basal epithelial cells, the animals also develop a synovitis, mimicking the changes seen in early-stage psoriatic arthritis.20Cook PW Brown JR Cornell KA Pittelkow MR Suprabasal expression of human amphiregulin in the epidermis of transgenic mice induces a severe, early-onset, psoriasis-like skin pathology: expression of amphiregulin in the basal epidermis is also associated with synovitis.Exp Dermatol. 2004; 13: 347-356Crossref PubMed Scopus (74) Google Scholar As a result of these past studies, amphiregulin has been suggested as a target for anti-psoriatic therapy. Based on this, we have in the present work examined a humanized anti-amphiregulin antibody for ability to suppress psoriatic hyperplasia in human skin transplanted to severe-combined immunodeficient (SCID) mice. Humanized antibodies have demonstrated therapeutic efficacy for a variety of conditions that include both infectious and immune-mediated diseases, as well as cancer. Antibody mediated cytokine-neutralization is an attractive strategy when elevated levels of the cytokine make a contribution to the pathology of the disease. This strategy has been unequivocally validated for anti-tumor necrosis factor-α agents in rheumatoid arthritis and Crohn's disease.21Palladino MA Bahjat FR Theodorakis EA Moldawer LL Anti-TNF-alpha therapies: the next generation.Nat Rev Drug Discov. 2003; 2: 736-746Crossref PubMed Scopus (507) Google Scholar Our results suggest that an anti-amphiregulin strategy may have therapeutic efficacy in human psoriasis. Human recombinant amphiregulin was obtained from R&D Systems (Minneapolis, MN). The recombinant protein was the 98-amino acid long form22Plowman GD Green JM McDonald VL Neubauer MG Disteche CM Todaro GJ Shoyab M The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity.Mol Cell Biol. 1990; 10: 1969-1981Crossref PubMed Scopus (320) Google Scholar expressed in Escherichia coli. BALB/c mice were immunized and boosted twice with the recombinant protein. Seven days after the second boost, serum titers against human recombinant amphiregulin were determined using a standard enzyme-linked immunosorbent assay (ELISA). Splenocytes were isolated from mice with the highest sera titer against human amphiregulin and fused with SP2/0 cells. The supernatants of the resulting hybridomas were screened by ELISA to identify those specific for human amphiregulin. Specificity of antibodies for human amphiregulin was confirmed by examining all supernatants positive for binding to amphiregulin for reactivity with EGF and heparin-binding EGF by ELISA. No supernatants exhibited reactivity with these related members of the EGF family. Reactivity of antibodies with native amphiregulin was confirmed by testing hybridoma supernatants on ELISA plates coated with goat anti-human amphiregulin antibody that had captured amphiregulin produced by phorbol ester-stimulated MCF-7 cells. All antibodies demonstrating reactivity with and specificity for native human amphiregulin were purified and assessed for the ability to inhibit amphiregulin-mediated proliferation of murine 3T3 fibroblasts and human keratinocytes. One antibody of the panel, designated PAR34 (a murine IgG2b, k), possessed potent amphiregulin-neutralizing activity and was chosen for humanization. PAR34 was humanized using techniques previously described.23Queen C Schneider WP Selick HE Payne PW Landolfi NF Duncan JF Avdalovic NM Levitt M Junghans RP Waldmann TA A humanized antibody that binds to the interleukin 2 receptor.Proc Natl Acad Sci USA. 1989; 186: 10029-10033Crossref Scopus (557) Google Scholar Briefly, the heavy and light chain variable regions of PAR34 were cloned and sequenced. The human heavy and light V region framework used as acceptors for the PAR34 complementarity determining regions (CDRs) were chosen based on sequence homology to the original murine antibody. The computer programs ABMOD and ENCAD24Levitt M Molecular dynamics of native protein. I. Computer simulation of trajectories.J Mol Biol. 1983; 168: 595-617Crossref PubMed Scopus (291) Google Scholar were used to construct a molecular model of the variable regions. Amino acids in the humanized V regions predicted to have contact with complementarity determining regions (CDRs) were substituted with the corresponding residues of PAR34. The amino acids in the humanized V region that were found to be rare in the same V region subgroup were changed to the consensus amino acids to eliminate potential immunogenicity. The humanized light and heavy chain variable region genes were constructed and expressed as described to result in a humanized version with the isotype of IgG1, κ.23Queen C Schneider WP Selick HE Payne PW Landolfi NF Duncan JF Avdalovic NM Levitt M Junghans RP Waldmann TA A humanized antibody that binds to the interleukin 2 receptor.Proc Natl Acad Sci USA. 1989; 186: 10029-10033Crossref Scopus (557) Google Scholar High-yielding transfectants were expanded and used to produce purified humanized PAR34 (HuPAR34). Functional comparison of PAR34 and HuPAR34 revealed that both murine PAR34 and HuPar34 bound specifically to human amphiregulin, but exhibited no reactivity to human EGF and heparin-binding EGF by ELISA. Biacore analysis of the murine and humanized versions revealed calculated kd values for PAR34 and HuPAR34 of 0.406 ± 0.082 nmol/L and 0.534 ± 0.050 nmol/L, respectively, indicating that the affinity of HuPAR34 to amphiregulin is ∼1.3-fold lower than that of PAR34, well within the twofold to threefold difference commonly seen on humanization. Comparison of the antibody's capacity to inhibit amphiregulin-mediated proliferation of murine 3T3 cells indicated a similar difference with PAR34 exhibiting an IC50 of 25 ng/ml, while HuPAR34 displayed an IC50 of 55 ng/ml. Thus the humanized version of PAR34 (HuPAR34) retained the amphiregulin-neutralizing activity of the original murine antibody and all further studies involved only HuPAR34. MSL-109 (human IgG1, κ), a human antibody to glycoprotein H of cytomegalovirus25Drobyski WR Gottlieb M Carrigan D Ostberg L Grebenau M Schran H Magid P Ehrlich P Nadler PI Ash RC Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients.Transplantation. 1991; 51: 1190-1196Crossref PubMed Scopus (28) Google Scholar was used as an isotype-matched control antibody for the anti-amphiregulin. Monoclonal antibodies to human CD3 (T-cell marker) and human von Willebrand factor (endothelial cell marker) were obtained from DAKO (Carpinteria, CA) and used for immunostaining of transplanted tissue. Six-mm punch biopsies of psoriatic lesional skin (four biopsies per volunteer) were obtained from four individuals with active psoriasis on the trunk or hip. None of the tissue donors were on therapy at the time of biopsy and none had been on systemic therapy for a period of at least 6 months. Six-mm punch biopsies of skin were also obtained from four nonpsoriatic volunteers (four biopsies per volunteer). The use of human tissue in this study was approved by the University of Michigan Institutional Review Board, and biopsies were obtained after receiving written informed consent from the skin donors. SCID mice (CB-17 strain; Taconic Farms Inc., Germantown, NY) were used as tissue recipients. One 6-mm punch biopsy was transplanted onto the dorsal surface of a recipient mouse as described previously.26Zeigler M Chi Y Tumas DB Bodary S Tang H Varani J Anti-CD11a ameliorates disease in the human psoriatic skin-SCID mouse transplant model: comparison of antibody to CD11a with cyclosporin A and clobetasol propionate.Lab Invest. 2001; 81: 1253-1261Crossref PubMed Scopus (40) Google Scholar, 27Ellis CN Varani J Fisher GJ Pershadsingh HA Benson SC Kurtz TW Troglitazone improves psoriasis and normalizes models of proliferative skin disease.Arch Dermatol. 2000; 136: 609-615Crossref PubMed Google Scholar Briefly, after mice were anesthetized, skin from the dorsal region was shaved. Mouse skin was surgically removed to size and replaced with the human tissue. The human tissue was secured to the back of the mice with absorbable sutures (4-0 Dexon S; Davis-Geck, Manati, Puerto Rico). The transplants were then bandaged with Xeroform petrolatum dressing (Kendall Co., Mansfield, MA) for 3 to 4 days. The animals were maintained in a pathogen-free environment throughout the preparation and treatment phases. Treatment was initiated 1 to 2 weeks after transplantation, depending on how rapidly the tissue healed. The human skin-SCID mouse transplant model has been used previously to study the pathophysiology of psoriasis28Nickoloff BJ Kunkel SL Burdick M Strieter RM Severe combined immunodeficiency mouse and human psoriatic skin chimeras. Validation of a new animal model.Am J Pathol. 1995; l46: 580-588Google Scholar, 29Wrone-Smith T Nickoloff BJ Dermal injection of immunocytes induces psoriasis.J Clin Invest. 1996; 98: 1878-1887Crossref PubMed Scopus (387) Google Scholar, 30Gilhar A David M Ullmann Y Berkutski T Kalish RS T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.J Invest Dermatol. 1997; 109: 283-288Crossref PubMed Scopus (117) Google Scholar, 31Dam TM Kang S Nickoloff BJ Voorhees JJ 1α25-dihydroxycholecalciferol and cyclosporin suppress induction and promote resolution of psoriasis in human skin grafts transplanted on to SCID mice.J Invest Dermatol. 1999; 113: 1082-1089Crossref PubMed Scopus (53) Google Scholar and to assess potential anti-psoriatic agents.26Zeigler M Chi Y Tumas DB Bodary S Tang H Varani J Anti-CD11a ameliorates disease in the human psoriatic skin-SCID mouse transplant model: comparison of antibody to CD11a with cyclosporin A and clobetasol propionate.Lab Invest. 2001; 81: 1253-1261Crossref PubMed Scopus (40) Google Scholar, 27Ellis CN Varani J Fisher GJ Pershadsingh HA Benson SC Kurtz TW Troglitazone improves psoriasis and normalizes models of proliferative skin disease.Arch Dermatol. 2000; 136: 609-615Crossref PubMed Google Scholar, 31Dam TM Kang S Nickoloff BJ Voorhees JJ 1α25-dihydroxycholecalciferol and cyclosporin suppress induction and promote resolution of psoriasis in human skin grafts transplanted on to SCID mice.J Invest Dermatol. 1999; 113: 1082-1089Crossref PubMed Scopus (53) Google Scholar Normal human skin and psoriatic lesional plaque skin transplanted onto SCID mice were treated with humanized anti-amphiregulin or the irrelevant control antibody (MSL-109). Briefly, after allowing the tissue to heal (1 to 2 weeks), mice were treated by an intraperitoneal injection of 200 μg of antibody (∼10 mg/kg) per animal every 3 days for 28 days. At the end of the treatment period, animals were sacrificed. The transplanted skin with a small amount of surrounding mouse skin was removed and fixed in 10% buffered formalin. The skin was examined histologically after staining with hematoxylin and eosin. Tissue sections were visualized by light microscopy at ×200 magnification. Epidermal area of each tissue section was captured in equal segments (normally three to four segments across a typical tissue section) and epidermal thickness in each area was assessed at four to five points. From these values, mean epidermal thickness was determined in a blinded manner. Before transplantation, a small piece of donor tissue was fixed in 10% buffered formalin and used for zero-time assessment of epidermal thickness. In addition to quantitative evaluation, skin grafts were also evaluated histologically for characteristic features of psoriasis including epidermal hyperplasia and rete peg formation. Tissue sections were also stained with an antibody to human CD3 to identify T cells in the tissue and with an antibody to human von Willebrand factor to identify capillaries. Staining was performed by the immunoperoxidase method using avidin-biotin complex staining as described previously.32Varani J Hattori Y Chi Y Schmidt T Perone P Zeigler ME Fader DJ Johnson TM Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin.Br J Cancer. 2000; 82: 657-665Crossref PubMed Scopus (75) Google Scholar Epidermal keratinocytes and dermal fibroblasts were isolated from normal human skin as described previously.33Varani J Perone P Griffiths CEM Inman D Fligiel SEG Voorhees JJ All-trans retinoic acid stimulates events in organ-cultured skin that underlie repair.J Clin Invest. 1994; 94: 1747-1753Crossref PubMed Google Scholar Keratinocytes were grown in keratinocyte growth medium, obtained from Cambrex (Walkersville, MD). Keratinocyte growth medium is a serum-free, low-Ca2+ (0.15 mmol/L) variant of MCDB-153 medium supplemented with EGF, insulin, and pituitary extract as growth supplements. Fibroblasts were grown in Dulbecco's minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). Cells were grown at 37°C in an atmosphere of 5% CO2 and 95% air, and used at passages 3 to 4. HuPAR34 and the control antibody (MSL-109) were examined for ability to inhibit keratinocyte proliferation in monolayer culture. Briefly, cells were plated in keratinocyte growth medium at 4 × 104 cells per well in wells of a 24-well dish. After allowing the cells to attach, the culture medium was removed and the cells were washed two times and then incubated in keratinocyte basal medium (Cambrex). Keratinocyte basal medium consists of the same basal medium as keratinocyte growth medium but is not supplemented with growth factors. Antibodies were added at the desired concentrations (5 to 50 μg/ml) and the cells incubated for 2 days. At the end of the incubation period, the cells were harvested with trypsin/ethylenediamine tetraacetic acid and counted. The assay conditions used were similar to those described previously34Pittelkow MR Cook PW Shipley GD Derynck R Coffey Jr, RJ Autonomous growth of human keratinocytes requires epidermal growth factor receptor occupancy.Cell Growth Differ. 1993; 4: 513-521PubMed Google Scholar, 35Cook PW Mattox PA Keeble WW Pittelkow MR Plowman GD Shoyab M Adelman JP Shipley GD A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.Mol Cell Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (207) Google Scholar and designed to allow autocrine stimulation of growth to occur. Amphiregulin is thought to be the major autocrine factor driving keratinocyte proliferation under these conditions.16Piepkorn M Pittelkow MR Cook PW Autocrine regulation of keratinocytes: the emerging role of heparin-binding, epidermal growth factor-related growth factors.J Invest Dermatol. 1998; 111: 715-721Crossref PubMed Scopus (121) Google Scholar, 17Piepkorn M Predd H Underwood R Cook P Proliferation-differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes.Arch Dermatol Res. 2003; 295: 93-101Crossref PubMed Scopus (63) Google Scholar, 35Cook PW Mattox PA Keeble WW Pittelkow MR Plowman GD Shoyab M Adelman JP Shipley GD A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.Mol Cell Biol. 1991; 11: 2547-2557Crossref PubMed Scopus (207) Google Scholar, 36Cook PW Pittelkow MP Shipley GD Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine and paracrine mitogenic factors.J Cell Physiol. 1991; 146: 277-289Crossref PubMed Scopus (90) Google Scholar Fibroblast proliferation was examined in the same manner except that Dulbecco's minimal essential medium-fetal bovine serum was used as plating medium and the Ca2+ concentration was increased to 1.4 mmol/L in the (keratinocyte basal medium) assay medium. Before harvest, fibroblast culture fluid was removed from each well and saved for assessment of type I procollagen (see below). Fibroblast culture fluids were also assayed for type I procollagen by ELISA (Pan Vera Corp., Madison, WI) as described previously.37Varani J Warner RL Mehrnaz G-K Phan SH Kang S Chung JH Wang ZQ Datta SC Fisher GJ Voorhees JJ Vitamin A antagonizes decreased cell growth and elevated collagen-degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.J Invest Dermatol. 2000; 114: 480-486Crossref PubMed Scopus (511) Google Scholar Differences between groups in experiments with two groups were analyzed using the Student's t-test. Multiple group experiments were analyzed for statistical significance by analysis of variance followed by paired-group comparisons. In the first series of studies, four psoriatic skin transplants were performed. Four mice were transplanted with skin from each volunteer. Two mice from each transplant were subsequently treated with the anti-amphiregulin antibody and two from each transplant were treated with the control antibody. Consistent with previous findings,26Zeigler M Chi Y Tumas DB Bodary S Tang H Varani J Anti-CD11a ameliorates disease in the human psoriatic skin-SCID mouse transplant model: comparison of antibody to CD11a with cyclosporin A and clobetasol propionate.Lab Invest. 2001; 81: 1253-1261Crossref PubMed Scopus (40) Google Scholar, 27Ellis CN Varani J Fisher GJ Pershadsingh HA Benson SC Kurtz TW Troglitazone improves psoriasis and normalizes models of proliferative skin disease.Arch Dermatol. 2000; 136: 609-615Crossref PubMed Google Scholar, 28Nickoloff BJ Kunkel SL Burdick M Strieter RM Severe combined immunodeficiency mouse and human psoriatic skin chimeras. Validation of a new animal model.Am J Pathol. 1995; l46: 580-588Google Scholar, 30Gilhar A David M Ullmann Y Berkutski T Kalish RS T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.J Invest Dermatol. 1997; 109: 283-288Crossref PubMed Scopus (117) Google Scholar, 31Dam TM Kang S Nickoloff BJ Voorhees JJ 1α25-dihydroxycholecalciferol and cyclosporin suppress induction and promote resolution of psoriasis in human skin grafts transplanted on to SCID mice.J Invest Dermatol. 1999; 113: 1082-1089Crossref PubMed Scopus (53) Google Scholar the psoriatic skin maintained a hyperplastic phenotype through the period after transplantation and during treatment. Treatment of the transplant-bearing animals with the humanized anti-amphiregulin antibody, but not with the isotype-matched control antibody, suppressed the hyperproliferative conditions in the transplanted skin. The top panels of Figure 1 demonstrate histological features seen in transplanted psoriatic skin after treatment with either the anti-amphiregulin antibody (Figure 1; A, C, and E) or the isotype-matched control (Figure 1; B, D, and F). The bottom panel provides a quantitative comparison of epidermal thickness between the two groups. In parallel studies, skin from four nonpsoriatic volunteers was transplanted to SCID mice and treated with the anti-amphiregulin and control a
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