Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues.

The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein-ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled µ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.

Predicting the segregation profile of the Pt25Rh75(100) surface from first-principles.

The segregation profile of the Pt(25)Rh(75)(100) surface is studied by the combination of density functional theory calculations with the cluster-expansion method and Monte Carlo simulations. We construct the stability diagram for the surface layers, which allows the prediction of the most stable atomic configuration for a given average concentration in those layers. On this basis, we apply the cluster-expansion Hamiltonian in grand-canonical Monte Carlo simulations for the prediction of the temperature-dependent concentration profile. The experimentally found enrichment of Pt in the top layer and depletion in the second layer is nicely confirmed by the calculations.

Pan-neurotrophin receptor p75NTR expression is strongly induced in lesional atopic mast cells.

BACKGROUND: Neurotrophins such as nerve growth factor or brain-derived neurotrophic factor influence neuronal proliferation and differentiation via the low-affinity pan-neurotrophin receptor p75NTR that may play a pivotal role in linking the immune with the nervous system. Because the precise regulation of p75NTR gene transcription in mast cells under states of allergic inflammation has not been investigated in detail so far, the present studies assessed the gene regulation and expression of this receptor. METHODS: Transcriptional expression of p75NTR in human skin was studied in isolated cutaneous cells by means of RT-PCR. In situ lesional mast cell p75NTR expression was analysed by immunohistochemistry. RESULTS: The p75NTR mRNA expression was found in isolated human skin mast cells and keratinocytes. Lower mRNA levels were present in fibroblasts and melanocytes but no transcripts were found in endothelial cells. The p75NTR protein expression was found in situ in lesional and non-lesional mast cells. A significantly increased expression of p75NTR protein was found in atopic dermatitis lesional mast cells when compared with control mast cell expression (P<0.05). CONCLUSION: The demonstration of an increased level of p75NTR gene transcription in lesional mast cells points to an induction of low-affinity neurotrophin receptor sensitivity of mast cells under states of allergic inflammation. Topically administered neurotrophin receptor-modulating compounds may act as anti-inflammatory mediators in cutaneous allergic inflammation.

Cell type-specific regulation of brain-derived neurotrophic factor in states of allergic inflammation.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is a molecule influencing neuronal proliferation and differentiation. In states of allergy, it may orchestrate inflammatory changes by linking the immune system with the nervous system. Because the precise regulation of gene transcription in mast cells MCs is not clear, the present studies assessed the gene regulation of BDNF in this inflammatory cell type. METHODS: Transcriptional expression of BDNF in human skin was studied in isolated cells using RT-PCR. In situ lesional MC BDNF protein expression was analysed by immunohistochemistry and related to the differential staining of MCs and functional effects of BDNF on HaCaT keratinocytes. RESULTS: BDNF mRNA expression was found in isolated human skin MCs, keratinocytes, and fibroblasts. Also, low levels were found in endothelial cells and melanocytes. BDNF protein expression was found in situ in lesional and non-lesional MCs. A significantly decreased expression of BDNF protein was found in atopic dermatitis lesional MCs when compared with control MC expression. Functional in vitro experiments demonstrated that a decrease in BDNF stimulation led to increased secretion rates for stem cell factor and IL-8 in HaCaT keratinocytes. CONCLUSION: The demonstration of a decreased level of BDNF gene transcription in lesional MCs points to a differential regulation of MC-released neutrotrophins in cutaneous allergic inflammation. Topically administered neurotrophin receptor-modulating compounds should be receptor target specific and not universally acting in diseases such as atopic dermatitis or allergic asthma.

Neurotrophins act as neuroendocrine regulators of skin homeostasis in health and disease.

Neurotrophins regulate cutaneous innervation, act as growth and motility factors on structural skin cells such as keratinocytes and fibroblasts, modulate cutaneous immune function and even serve as stress mediators in skin biology. The multilayered neurotrophin interaction with skin biology through high affinity specific tyrosinekinase receptors and the Janus-faced p75 receptor, which depending on ligand and co-receptor expression can serve as a low-affinity pan-neurotrophin receptor or a high affinity proneurotrophin receptor, guaranties this neuroendocrine peptide family a central position in the control of skin homeostasis in health and disease. It is a challenging task for future research efforts to integrate our knowledge on differential neurotrophin expression patterns and signaling pathways into complex concepts of neuroendocrine tissue remodeling and pathogenetic processes. In addition, we need to improve our understanding of the role of neurotrophin processing enzymes, associated co-receptors and intracellular adaptor molecules in specific cutaneous cell populations to design precise interaction tools for research and treatment. Such tools will allow us to utilize this ancient growth factor family in the management of neurotrophin responsive pathogenetic pathways and cutaneous diseases such as neurogenic inflammation, peripheral nerve degeneration, wound healing, atopic dermatitis or psoriasis.

Gene expression and regulation of transcription factor activator protein-2 alpha in human mast cells.

BACKGROUND: The transcription factor activator protein (AP)-2 regulates cell-type specific gene expression during development and differentiation, but its role in mast cell development has so far not been explored. METHODS: Gene expression and regulation of AP2 was assessed in normal skin, diseases with increased mast cell numbers, and in vitro models of mast cell differentiation. RESULTS: AP-2alpha-protein was not detectable in normal skin but in mastocytoma lesional mast cells. AP-2alpha-mRNA and -protein were also detected in leukemic mast cells (HMC-1), in the adherent fraction of peripheral blood (PBMC) and umbilical cord blood mononuclear cells (CBMC), and AP-2alpha-mRNA at low levels in isolated-purified mast cells. During culture with fibroblast supernatants or SCF, AP-2alpha-mRNA was de novo expressed in KU812-cells, maintained at about the same level in PBMC and CBMC, and upregulated in HMC-1-cells. On extended culture, a down-regulation was noted at mRNA and/or protein levels. In contrast, tryptase expression increased in all cells throughout culture, as did c-Kit in normal cells, whereas in both leukemic cell lines, c-Kit was maintained unchanged at about the same level. CONCLUSIONS: These findings suggest a continuous activation of AP-2alpha in mastocytomas and mast cell leukemia and its transient upregulation during c-Kit dependent early steps of normal mast cell differentiation.

Mast cells and vasculature in atopic dermatitis--potential stimulus of neoangiogenesis.

BACKGROUND: Atopic dermatitis skin lesions are characterized by inflammatory changes and epithelial hyperplasia requiring angiogenesis. As mast cells may participate in this process via bidirectional secretion of tissue-damaging enzymes and pro-angiogenic factors, the present study aimed to assess the occurrence and possible function of mast cells in the papillary dermis and in epidermal layers of atopic dermatitis lesions. METHODS: Semi-thin and serial sections in combination with immunohistochemistry, histochemistry and proliferating cell nuclear antigen (PCNA)-activity assays were used and related to epidermal thickness and targeted gene expression studies. RESULTS: Mast cells were located in the papillary dermis and migrated through the basal lamina into the epidermis of atopic dermatitis lesions. An increased PCNA-activity in cells of superficial epidermal layers indicated an activation of keratinocytes and stimulation of endothelial growth. Only approximately 30% of the papillary mast cells stained with the tryptase were toluidin-blue-positive, and approximately 80% were chymase positive. A high number of mast cells expressed c-kit. Most papillary and epidermal mast cells were localized close to endothelial cells. Vascular expression of endoglin (CD105) demonstrated neoangiogenic processes. Mast cells stimulation led to the expression of proangiogenic factors. Also, gene expression of tissue-damaging factors such as matrix metalloproteinases was increased. CONCLUSIONS: These data suggest that in atopic dermatitis, mast cells are abundantly localized close to and within the epidermis where they may stimulate neoangiogenesis. Via the new vessels, inflammatory cells, together with complement components and antibodies, can be transported to the epidermis to aid in the defense against environmental antigens and to maintain chronic inflammation.

Aspirin-sensitive rhinitis-associated changes in upper airway innervation.

Aspirin-sensitive rhinitis is the manifestation of aspirin intolerance in the upper respiratory tract. The disease represents a pseudoallergy against aspirin or related nonsteroidal anti-inflammatory drugs. As a major immunomodulatory role for airway innervation has been proposed in airway inflammatory diseases, the present study assessed changes in human nasal mucosa innervation in patients with aspirin-sensitive rhinitis in comparison to a control group. Immunohistochemistry for protein gene product 9.5, tachykinins, calcitonin gene-related peptide, vasoactive intestinal peptide (VIP) and neuropeptide tyrosine was performed on cryostats sections of nasal mucosa and neuropeptide containing nerves were examined independently using a score grading. In comparison to the control, the aspirin-sensitive rhinitis group had a significant increase of VIP-like immunoreactivity in mucosal nerve fibres. In contrast to constant numbers of mast cells, highly significant increases in the numbers of eosinophils were found in the group of aspirin-sensitive rhinitis patients. In summary, the present quantification of neuropeptide-immunoreactivity of mucosal nerves demonstrated differences in the human nasal mucosa innervation between nonrhinitic and aspirin-sensitive rhinitic individuals. These differences may reflect a pathophysiological role of upper airway innervation in pseudoallergic reactions.

Effect of granulocyte macrophage colony-stimulating factor in a patient with benign systemic mastocytosis.

We report the in vitro and in vivo effects of granulocyte macrophage colony stimulating factor (GM-CSF), a known inhibitor of in vitro mast cell differentiation, in a patient with benign, adult-onset systemic mastocytosis. In vitro effects of GM-CSF on bone marrow cultures before the start of treatment showed a marked inhibition of mast cell marker expression [tryptase, Kit, and high-affinity IgE receptor (FcepsilonRIalpha)] at both protein and mRNA levels. Therefore, the patient was treated with daily injections of GM-CSF for 10 weeks. After an initial improvement, increasing worsening of clinical symptoms was noted, and the patient refused further treatment. Lesional skin biopsies showed an increase of toluidine blue-positive mast cells, compared with uninvolved skin, with further significant increase after treatment. Similar results were obtained on staining for mast cell-specific tryptase and Kit, as well as for CD1a and FcepsilonRIalpha. These findings show that GM-CSF inhibits human bone marrow mast cell differentiation in vitro, and also in mastocytosis. However, GM-CSF apparently enhances recruitment of mast cell as well as dendritic cell precursors into the tissue during systemic treatment. These findings and the observed adverse clinical effects in the present patient make it unlikely that GM-CSF monotherapy will be beneficial for the treatment of mastocytosis.

Human skin mast cells rapidly release preformed and newly generated TNF-alpha and IL-8 following stimulation with anti-IgE and other secretagogues.

Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF-alpha and IL-8 as well as the pro-allergic cytokines IL-4, IL-5 and IL-13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE-dependent or IgE-independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF-alpha and IL-8 following activation with either anti-IgE, SCF, substance P, compound 48/80 or A23187. This release was dose- and time-dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF-alpha and IL-8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL-4, IL-5 or IL-13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro-inflammatory cytokines like TNF-alpha and IL-8 following IgE-dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL-4, IL-5 or IL-13 in these settings.

The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) is a potent inhibitor of mediator release from human dermal mast cells and peripheral blood basophils.

BACKGROUND: The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) has recently been developed as a novel and cell-selective inhibitor of inflammatory cytokine secretion; it has fewer adverse effects than currently available drugs. OBJECTIVE: In this study, we investigated the capacity of pimecrolimus to directly inhibit in vitro mediator release from human skin mast cells and basophils. METHODS: Purified cutaneous mast cells or basophil-containing peripheral blood leukocytes were obtained from healthy human donors and preincubated with pimecrolimus (0.1 nmol/L to 1 micromol/L) in the absence or presence of its specific antagonist (rapamycin), cyclosporin A (100 nmol/L to 1 micromol/L), or dexamethasone (1 micromol/L) and then stimulated with anti-IgE or with calcium ionophore A23187 plus phorbol myristate acetate. Cell supernatants were kept for analysis of histamine, tryptase, LTC4, and TNF-alpha. RESULTS: Pimecrolimus caused a strong and dose-dependent inhibition of anti-IgE--induced release of histamine from mast cells and basophils (maximally 73% and 82%, respectively, at 500 nmol/L pimecrolimus) and of mast cell tryptase (maximally 75%) and a less pronounced inhibition of LTC4 (maximally 32%) and of calcium ionophore plus phorbol myristate acetate--induced mast cell TNF-alpha release (90% maximum at 100 nmol/L pimecrolimus). In contrast, inhibition achieved during mast cell histamine release was maximally 60% with cyclosporin A and only 28% with dexamethasone. CONCLUSION: These data demonstrate a marked inhibitory capacity of pimecrolimus on mediator release from human mast cells and basophils with a potency exceeding that of cyclosporin A and dexamethasone. Pimecrolimus might thus be expected to be effective in the treatment of mast cell-- and basophil-dependent diseases.

GM-CSF downmodulates c-kit, Fc(epsilon)RI(alpha) and GM-CSF receptor expression as well as histamine and tryptase levels in cultured human mast cells.

GM-CSF is known primarily as a hematopoietic growth factor, but it has also been shown to inhibit mast cell differentiation in vitro. In order elucidate the mechanisms involved, we investigated the effects of GM-CSF in vitro on the differentiation of human leukemic mast cells (HMC-1 cells) and normal cord blood-derived mast cells (CBMC) under the influence of SCF, NGF, and fibroblast supernatant (FS). Under all culture conditions, GM-CSF induced a dose- and time-dependent reduction in intracellular histamine levels, tryptase activity, and numbers of cells immunoreactive for c-Kit and FcepsilonRIalpha. This effect leveled off between 10-100 ng/ml and after 4 days of culture. There was an associated decrease in mRNA expression for c-kit, FcepsilonRIalpha and tryptase. In contrast, no significant changes in the expression of the NGF receptor TrkA were noted under the same conditions. The GM-CSF receptor was found in HMC-1 cells and CBMC at both the mRNA and protein levels, but its expression decreased during culture with FS, and even more markedly during culture with GM-CSF. GM-CSF thus selectively inhibits in vitro induction and/or upregulation of all major mast cell characteristics in HMC-1 cells and CBMC irrespective of the growth factors present, and a concomitant downregulation of GM-CSF receptors can counteract these effects. GM-CSF may therefore function as a regulatory factor in mast cell growth and differentiation under normal and pathological conditions.

Expression of mast cell growth modulating and chemotactic factors and their receptors in human cutaneous scars.

In order to explore possible mechanisms involved in the previously documented turnover of mast cell subpopulations in human cutaneous scars, we have examined selected factors known to stimulate and/or modulate mast cell hyperplasia (SCF, NGF, TGFbeta1, GM-CSF) and their receptors in human cutaneous scar tissue. On immunohistochemistry, numbers of SCF- and TGFbeta1-positive cells were significantly increased in the epidermis and throughout the dermis in scars (n = 27) of varying ages (4-369 d old), compared with normal skin (n = 12). Furthermore, TRbetaRI, II, and the NGF-p75 receptors were significantly increased in the epidermis, TRbetaRI and NGF-TrkA throughout the dermis, and TRbetaRII, NGF-p75, and GM-CSFR only in the mid- and lower dermis of scars. NGF and GM-CSF expression was in contrast scarce and weak, with no differences between normal skin and scars. In tissue extracts, mRNA levels of SCF, TGFbeta1, TRbetaI and II, and both NGF-receptors, but not GM-CSFR, were significantly increased as well. TRbetaI and II were identified in up to 90% and 83%, respectively, of isolated normal skin mast cells on flow cytometry, and GM-CSFR and NGFR-p75 were identified on 70% and 73%, respectively, of avidin-positive normal mast cells on double immunofluorescence microscopy. As described before for the SCF receptor KIT, GM-CSFR and NGFR-p75 were partly or entirely downregulated on avidin-positive mast cells in scars. The marked upregulation of TGFbeta1, its type I and II receptors, and SCF suggest that these factors play a major role in the orchestration of mast cell increase in human cutaneous scars whereas the role of NGF and GM-CSF is less clear, despite the significant upregulation of their receptors.

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.

Inhibition of cytokine secretion from human leukemic mast cells and basophils by H1- and H2-receptor antagonists.

H1-type antihistamines have recently been reported to inhibit cytokine secretion from human and murine mast cells and basophils. In order to confirm and expand these studies, we have compared several H1-blockers and the H2-blocker ranitidine for their effect on TNF-alpha, IL-3, 6, 8 and GM-CSF release from human leukemic mast (HMC-1) and basophilic (KU812) cells, compared to dexamethasone. Cells were stimulated for 24 h with phorbol myristate acetate (25 ng/ml) and calcium ionophore A 23187 (2.5x10(-7) M) alone or with the drugs added at 10(-4) to 10(-15) M, and production of cytokines was measured by ELISA. All antihistamines caused a dose-dependent inhibition of TNF-alpha release from HMC-1 cells, with maximal effects at 10(-12) M for azelastine, 10(-9) M for loratadine and cetirizine, and 10(-8) M for ranitidine. The inhibitory potency of H1-blockers on cytokines from HMC-1 cells was TNF-alpha >IL-8> or =IL-6> or =IL-3, with no significant effects on GM-CSF. In KU812 cells which failed to secrete TNF-alpha and GM-CSF, the sequence was IL-6 >IL-8 after preincubation. Dexamethasone inhibited all cytokines, but ranitidine only TNF-alpha and IL-3. Antihistamines had no effect on calcium flux in resting or stimulated cells. At the mRNA level, inhibition was only seen with KU812 cells and IL-8 in the presence of azelastine at 10-(10) M. These data show thus distinct inhibitory patterns for different antihistamines during cytokine production from human mast cells and basophils which may contribute to the anti-inflammatory effects of these drugs during treatment of allergic diseases.

Expression of SCF splice variants in human melanocytes and melanoma cell lines: potential prognostic implications.

Stem cell factor (SCF), the ligand for c-Kit, is known to regulate developmental and functional processes of haematopoietic stem cells, mast cells and melanocytes. Two different splice variants form predominantly soluble (sSCF or SCF-1) and in addition some membrane-bound SCF (mSCF or SCF-2). In order to explore the prognostic significance of these molecules in melanoma, total SCF, SCF splice variants and c-Kit expression were studied in normal skin melanocytes and in 11 different melanoma cell lines, using reverse transcription polymerase chain reaction, immunocytochemistry and enzyme-linked immunosorbent assay. Nine of the 11 melanoma cell lines expressed SCF-1 mRNA, only two of them SCF-2, and these two also SCF-1. Coexpression of both SCF-1 and c-Kit was noted in five cell lines, and only one cell line as well as normal melanocytes expressed both SCF-1 and SCF-2 as well as c-Kit. Corresponding results were obtained on immunocytochemical staining. Of three exemplary melanoma cell lines studied, two expressing SCF mRNA also released SCF spontaneously and on stimulation, whereas the line lacking SCF and c-kit mRNA (SK-Mel-23) failed to do so. These data demonstrate thus that melanoma cell lines, particularly those known to metastasize in vivo, lose the ability to express SCF-2 mRNA, suggesting that this molecule may serve, next to c-Kit, as a prognostic marker for malignant melanoma.

Intercellular adhesion molecule-1 and hair follicle regression.

Although the intercellular adhesion molecule-1 (ICAM-1) is recognized for its pivotal role in inflammation and immune responses, its role in developmental systems, such as the cyclic growth (anagen) and regression (catagen) of the hair follicle, remains to be explored. Here we demonstrate that ICAM-1 expression in murine skin is even more widespread and more developmentally regulated than was previously believed. In addition to endothelial cells, selected epidermal and follicular keratinocyte subpopulations, as well as interfollicular fibroblasts, express ICAM-1. Murine hair follicles express ICAM-1 only late during morphogenesis. Thereafter, morphologically identical follicles markedly differ in their ICAM-1 expression patterns, which become strikingly hair cycle-dependent in both intra- and extrafollicular skin compartments. Minimal ICAM-1 and leukocyte function-associated (LFA-1) protein and mRNA expression is observed during early anagen and maximal expression during late anagen and catagen. Keratinocytes of the distal outer root sheath, fibroblasts of the perifollicular connective tissue sheath, and perifollicular blood vessels exhibit maximal ICAM-1 immunoreactivity during catagen, which corresponds to changes of LFA-1 expression on perifollicular macrophages. Finally, ICAM-1-deficient mice display significant catagen acceleration compared to wild-type controls. Therefore, ICAM-1 upregulation is not limited to pathological situations but is also important for skin and hair follicle remodeling. Collectively, this suggests a new and apparently nonimmunological function for ICAM-1-related signaling in cutaneous biology.

Nerve growth factor-beta induces mast-cell marker expression during in vitro culture of human umbilical cord blood cells.

Nerve growth factor-beta (NGF) is known as a growth factor for human basophils and murine mast cells and has recently been shown to also up-regulate mast cell characteristics in human leukaemic mast cells. We have examined here the effect of NGF on the differentiation of normal human mast cells from cord blood progenitors during culture with stem cell factor (SCF), NGF alone or in combination, or fibroblast supernatants. All these supplements induced mast cell immunoreactivity against tryptase, c-Kit and FcepsilonRIalpha, but none of the cells reacted against the basophil specific antibody 2D7 before or during culture. Intracellular tryptase activity increased as well, with maximal levels on combined culture with SCF and NGF. On reverse transcription-polymerase chain reaction (RT-PCR), cells lacked tryptase and chymase and expressed low levels of FcepsilonRI and c-Kit mRNA prior to culture, with marked up-regulation of FcepsilonRI and c-Kit, and with de novo expression of mast-cell specific alpha- and beta-tryptase by week 3, and of chymase by week 5. Only the TrkA and not the p75 NGF receptor was detected at m-RNA and protein level, and only the TrkA NGF receptor was up-regulated during NGF-driven culture. These findings show therefore that, like SCF, NGF is another growth factor that can induce and regulate human mast-cell development and differentiation.

New roles for glial cell line-derived neurotrophic factor and neurturin: involvement in hair cycle control.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and their receptors, GDNF family receptor alpha-1 (GFRalpha-1) and GDNF family receptor alpha-2 (GFRalpha-2), are critically important for kidney and nervous system development. However, their role in skin biology, specifically in hair growth control, is as yet unknown. We have studied expression and function of GDNF, neurturin, GFRalpha-1, and GFRalpha-2 in murine skin during the cyclic transformation of the hair follicle (HF) from its resting state (telogen) to active growth (anagen) and then through regression (catagen) back to telogen. GDNF protein and GFRalpha-1 messenger RNA are prominently expressed in telogen skin, which lacks NTN and GFRalpha-2 transcripts. Early anagen development is accompanied by a significant decline in the skin content of GDNF protein and GFRalpha-1 transcripts. During the anagen-catagen transition, GDNF, GFRalpha-1, NTN, and GFRalpha-2 transcripts reach maximal levels. Compared with wild-type controls, GFRalpha-1 (+/-) and GFRalpha-2 (-/-) knockout mice show a significantly accelerated catagen development. Furthermore, GDNF or NTN administration significantly retards HF regression in organ-cultured mouse skin. This suggests important, previously unrecognized roles for GDNF/GFRalpha-1 and NTN/GFRalpha-2 signaling in skin biology, specifically in the control of apoptosis-driven HF involution, and raises the possibility that GFRalpha-1/GFRalpha-2 agonists/antagonists might become exploitable for the treatment of hair growth disorders that are related to abnormalities in catagen development.

Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal-epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis-driven HF regression (catagen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild-type controls, transgenic mice overexpressing HGF/SF under the control of the MT-1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met-mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.-Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling.