Silvia Bulfone-Paus

Inhibition of keratinocyte apoptosis by IL-15 : a new parameter in the pathogenesis of psoriasis?

Keratinocytes (KC) are important source of and targets for several cytokines. Although KC express IL-15 mRNA, the functional effects of IL-15 on these epithelial cells remain to be dissected. Investigating primary human foreskin KC and HaCaT cells, we show here by semiquantitative RT-PCR and flow cytometric analysis that both translate IL-15 and IL-15R mRNA and express IL-15 and IL-15Rα protein on the cell surface, suggesting that human KC can employ IL-15 for juxtacrine signaling. While IL-15 exerted no significant effect on KC proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is in line with the recognized potent anti-apoptotic effects of IL-15. IL-2, whose receptor shares two components with the IL-15R, failed to inhibit KC apoptosis. Together with the role of IL-15 in sustaining chronic immune reactions, this invited the question of whether a reduction of KC apoptosis by IL-15 may be involved in the pathogenesis of psoriasis, a chronic hyperproliferative inflammatory skin disease characterized by abnormally low KC apoptosis in the epidermis. Remarkably, compared with nonlesional psoriatic skin and skin of healthy volunteers, lesional psoriatic epidermis showed high IL-15 protein expression in the epidermis and enhanced binding activity for IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC apoptosis deserves exploration as a novel therapeutic strategy in psoriasis management.

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apo‐ptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth‐modulatory function of BDNF, NT‐4, and their high‐affinity receptor, TrkB. The cutaneous expression of BDNF and NT‐4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis‐driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT‐4‐immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT‐4 knockout mice showed significant catagen retardation, whereas BDNF‐overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT‐4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT‐4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth‐modulatory drugs for the management of common hair growth disorders.—Botch‐karev, V. A., Botchkareva, N. V., Welker, P., Metz, M., Lewin, G. R., Subramaniam, A., Bulfone‐Paus, S., Hagen, E., Braun, A., Lommatzsch, M., Renz, H., Paus, R. A new role for neurotrophins: involvement of brain‐derived neurotrophic factor and neurotro‐phin‐4 in hair cycle control. FASEB J. 13, 395–410 (1999)

A New Role for Neurotrophin-3 : Involvement in the Regulation of Hair Follicle Regression (Catagen)

Nervous system and hair follicle epithelium share a common ectodermal origin, and some neurotrophins (NTs) can modulate keratinocyte proliferation and apoptosis. Therefore, it is reasonable to ask whether NTs are also involved in hair growth control. Here, we show that the expression of NT-3 and its high-affinity receptor, tyrosine kinase C, in the skin of C57BL/6 mice is strikingly hair cycle-dependent, with maximal transcript and protein expression seen during spontaneous hair follicle regression (catagen). During catagen, NT-3 and tyrosine kinase C are co-expressed by terminal deoxynucleotidyl transferase-mediated in situ nick end labeling-positive keratinocytes in the club hair and secondary germ. NT-3-overexpressing transgenic mice show precocious catagen development during the postnatal initiation of hair follicle cycling, whereas heterozygous NT-3 knockout (+/-) mice display a significant catagen retardation. Finally, NT-3 stimulates catagen development in organ culture of normal C57BL/6 mouse skin. These observations suggest that the hair follicle is both a source and target of NT-3 and that NT-3/tyrosine kinase C signaling is functionally important in the control of hair follicle regression. Therefore, tyrosine kinase C agonists and antagonists deserve systematic exploration for the management of hair growth disorders that are related to premature (alopecia/effluvium) or retarded catagen (hirsutism/hypertrichosis).

High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo

Backgroundu2002Hair loss following skin inflammation may in part be mediated by keratinocyte (KC) apoptosis. While the effects of different cytokines or other apoptosis stimulating agents such as interferon (IFN)‐γ or tumour necrosis factor (TNF)‐α on KC apoptosis in vitro have been addressed in several studies, little is known about the effects of proinflammatory cytokines on KC apoptosis in vivo.

IL-15-IgG2b fusion protein accelerates and enhances a Th2 but not a Th1 immune response in vivo, while IL-2-IgG2b fusion protein inhibits both

We have explored how IL‐15 influences Th1 or Th2 type immune response in vivo. Intraperitoneal application of an IL‐15‐IgG2b fusion protein (FP) to mice did neither significantly affect the footpad swelling nor the production of hemagglutinizing antibodies in a delayed type hypersensitivity reaction to sheep red blood cells. In contrast, in an established murine Th2 model of sensitization to ovalbumin (OVA), IL‐15‐IgG2b FP plus OVA sensitization resulted in massively accelerated and enhanced allergen‐specific IgE and IgG1 antibody production. In vitro, stimulation of spleen cells from OVA‐sensitized mice with OVA+IL‐15 or OVA+IL‐15‐IgG2b resulted in a significantly enhanced IgE production. IL‐4 secretion was significantly induced by IL‐15 but not by IL‐15‐IgG2b. An IL‐2‐IgG2b FP with the same Fc tail as the IL‐15‐IgG2b FP was used as control in both models. In striking contrast to the IL‐15‐IgG2b FP, IL‐2‐IgG2b significantly inhibited the Th2 type antibody production in vivo. The current study suggests that IL‐15‐IgG2b may be employed as a potent accelerator and enhancer of Th2 type immune responses in vivo, while IL‐2‐IgG2b can suppress the latter.

Inhibition of corneal allograft reaction by CTLA4-Ig

Abstract• Background: Activation of T cells requires both the interaction of T-cell receptor with major histocompatibility complex on the antigen-presenting cell and costimulatory signals, for instance the B7 antigens expressed on antigen-presenting cells and the CD28 molecule expressed on T cells. A recombinant fusion protein, CTLA4-Ig, has been produced that contains the extracellular domain of human CTLA4 fused to IgGl constant region and that binds the B7 molecule with high affinity. Blocking the CD28/B7 interaction with CTLA4-Ig inhibits T cell activation in vitro and in vivo. • Methods: We used CTLA4-Ig in a fully MHC-mismatched mouse keratoplasty model. The animals were divided into four groups: (1) no treatment, (2) intraperitoneal treatment with 130 μg CTLA4-Ig, (3) intraperitoneal treatment with 300 μg CTLA4-Ig, (4) subconjunctival treatment with 290 μg CTLA4-Ig. • Results: The allograft reaction occurred in untreated animals between days 12 and 16 (mean 13.5). While topical application of CTLA4-Ig seemed to shorten the graft survival (mean 11.6 days) and systemic application of 130 μg had no influence (mean 14.0), only intraperitoneal injection of 300 μg of CTLA4-Ig prolonged the survival of allografts (mean >20 days) (P<0.01). • Conclusion: CTLA4-Ig prolonged significantly the survival of corneal allografts in a fully MHC-mismatched mouse keratoplasty model, but the small antigen load of the corneal transplant and the anterior chamber-associated immune deviation (ACAID) may have a disadvantage to induce tolerance in this model of CTLA4-Ig therapy.

The effect of corticosteroid and cyclosporin A on murine corneal allograft rejection

Abstractu2002Background: The immunomodulatory T-helper type 1 (Th1) cytokine interferon-γ (IFN-γ) was measured in serum and cornea to ascertain its general contribution to corneal graft rejection and to establish a rational basis for the decision for or against systemic therapy. Methods: Eight groups of differently treated BALB/c (H-2d) mice received a C3H (H-2 k) corneal graft. There was one saline-treated control group and two groups that received intramuscular cyclosporin A (CsA) for 14 or 40. Three groups received systemic or topical, systemic plus topical corticosteroid treatment, which was combined with CsA in two further groups. To measure the IFN-γ level by enzyme-linked immunosorbent assay (ELISA), blood was taken by heart puncture and corneae were excised at the limbus. Results: Five days of systemic corticosteroid and 14 days of CsA had no significant effect on graft survival. A 40-day CsA treatment and a 40-day combined corticosteroid treatment significantly prolonged graft survival. An 80-day topical corticosteroid treatment produced additional prolongation. IFN-γ could not be detected (limit of detection 25 pg/ml) in any of the serum samples, while significantly increased amounts of IFN-γ were detected in the supernatants of the corneal tissue 13 or 14 days after allogeneic but not syngeneic corneal graft, corresponding to 9.5 pg, 5.1 pg and 1.8 pg per cornea. Conclusion: The detection of Th1 cytokines in the cornea but not the serum of mice at the time of allograft rejection is in accordance with the finding of long-lasting dose-dependent immunosuppression of topical steroids and the inefficacy of short-term systemic CsA and corticosteroids.

Structure of the Hodgkin's lymphoma-associated human CD30 gene and the influence of a microsatellite region on its expression in CD30(+) cell lines.

The CD30 antigen is a member of the tumor necrosis factor receptor (TNFR) family which is overexpressed on the surface of the tumor cells of Hodgkins lymphoma, anaplastic large cell lymphoma (ALCL), and embryonal carcinoma of the testis. In this study the entire cd30 gene which is more than 24000 bp long and organized in eight exons was characterized by analyzing cosmid and phage lambda clones from human placental libraries with long-range polymerase chain reaction (PCR) and sequencing. Differences to other genes of the TNFR family were detected in the region encoding the extracellular domain of the cd30 gene. In nearly all other TNFR genes, the coding region of each cysteine-rich repeat is interrupted by one intron, i.e., the 3-4 cysteine-rich repeats of these receptors are encoded by at least 4-5 exons, whereas the six cysteine-rich repeats of the cd30 gene are encoded by two exons, i.e., each of these exons encode three cysteine-rich repeats. In addition, we also found a genetic polymorphism of tetranucleotide ATCC-repeats in the 5 part of the CD30 promoter. This region was amplified by PCR from seven CD30 overexpressing human lymphoid cell lines and five human tissues with an absent or very low CD30 expression. The amplification products showed length differences of more than 550 bp. The number of the ATCC-repeats was higher in CD30(+) cell lines than in normal tissues. Comparison of the individual PCR products in reporter gene assays revealed that the CD30 promoter activity increased with the length of this polymorphic region up to eightfold. The data suggest that the number of ATCC-repeats in the 5 region of the CD30 promoter modulates the regulation of CD30 expression.

Expression of several members of the TNF‐ligand and receptor family on tonsillar lymphoid B cells

Although the expression patterns of the members of the tumour necrosis factor receptor and ligand families have extensively been studied by flow‐cytometry on stimulated peripheral blood mononuclear cells (PBMNC), little or no flow‐cytometric or immunohistological data exist about their expression in lymphoid tissue. According to the data obtained from stimulated PBMNC, several members of these molecule families (e.g. CD40 ligand [CD40L], CD30, CD27, hOX40) have been considered to be either T‐cell restricted or strongly T‐cell associated. The present study on samples from palatine tonsils revealed that most of these molecules are also expressed by tonsillar B cells. The additional analysis of the co‐expression of these molecules also disclosed the existence of CD40+/CD40L+ and CD27+/CD70+ (CD27L+) lymphoid cells in tonsillar tissue.

Prolongation of corneal allograft survival by an interleukin-2-immunoglobulin fusion protein in mice

Abstractu2002· Background: Interleukin 2 (IL2) production by activated T-helper cells leads to activation and proliferation of cytotoxic T cells. Recently, an IL2-IgG fusion protein was found to suppress cell-mediated and humoral immune responses in mice.u2002· Methods: We used the genetically engineered murine IL2-IgG2b fusion protein in a fully MHC-mismatched mouse keratoplasty model. The DTH reaction against sheep red blood cells was investigated as a measure of IL2-IgG2b-mediated immunosuppression. The animals were divided into three control groups (n≥6) [no treatment, subconjunctival (SQ) treatment with saline or mouse serum], two IL2 SQ-treated groups (14 μg or 140 μg), and four IL2-IgG2b-treated groups (14 μg, 140 μg or 280 μg SQ or 280 μg IP). · Results: Administration of 20 μg of IL2-IgG2b twice daily from the time of immunization until the time of challenge resulted in almost complete prevention of footpad swelling. The 140 μg SQ application of IL2 (allograft reaction on day 20.5 ± 4.04) and the 280 μg SQ (day 19.2 ± 2.48) or IP (day 19.7 ± 1.5) application of IL2-IgG2b fusion protein significantly prolonged the corneal graft survival in comparison to the untreated group (day 13.4 ± 1.35) (P<0.01) or saline control group (P<0.01) and the mouse-serum-treated group (day 14.7 ± 3.5) (P<0.05).u2002· Conclusion: Our results indicate that, at a total dose of 280 μg, the fusion protein IL2-IgG2b causes no detectable side effects and very effectively suppresses the immune response of the corneal allograft in mice. This fusion protein could prove useful in the treatment of allograft rejections and autoimmune diseases.