Sven Müller-Röver

The human hair follicle immune system: cellular composition and immune privilege.

The immunology of the hair follicle, its relationship with the ‘skin immune system’ and its role in hair diseases remain biologically intriguing and clinically important. In this study, we analysed the immunoreactivity patterns of 15 immunodermatological markers to determine the cellular composition and immune privilege of the human hair follicle immune system in anagen VI (growth phase). The most prominent cells located in or around the hair follicle were Langerhans cells, CD4+ or CD8+ T cells, macrophages and mast cells, whereas B cells, natural killer cells and γδ T cells were found very rarely. Langerhans cells (CD1a+, major histocompatibility complex, MHC class II+), and T cells (CD4+ or CD8+) were predominantly distributed in the distal hair follicle epithelium, whereas macrophages (CD68+, MHC class II+) and mast cells (Giemsa+) were located in the perifollicular connective tissue sheath. Transmission electron microscopy confirmed low numbers of immune cells in the proximal hair follicle epithelium, and very few macrophages and Langerhans cells were seen in the dermal papilla. Melanophages were observed in the connective tissue sheath and dermal papilla. MHC class I (HLA‐A, ‐B, ‐C) and β2‐microglobulin immunoreactivity was found on most skin cells, but was substantially reduced on isthmus keratinocytes and virtually absent in the proximal hair follicle epithelium. Apart from the absence of Fas ligand immunoreactivity, the sharply reduced numbers of T cells and Langerhans cells, and the virtual absence of MHC class I expression all suggest that the anagen proximal hair follicle constitutes an area of immune privilege within the hair follicle immune system, whose collapse may be crucial for the pathogenesis of alopecia areata.

‘Cyclic alopecia’ in Msx2 mutants: defects in hair cycling and hair shaft differentiation

Msx2-deficient mice exhibit progressive hair loss, starting at P14 and followed by successive cycles of wavelike regrowth and loss. During the hair cycle, Msx2 deficiency shortens anagen phase, but prolongs catagen and telogen. Msx2-deficient hair shafts are structurally abnormal. Molecular analyses suggest a Bmp4/Bmp2/Msx2/Foxn1 acidic hair keratin pathway is involved. These structurally abnormal hairs are easily dislodged in catagen implying a precocious exogen. Deficiency in Msx2 helps to reveal the distinctive skin domains on the same mouse. Each domain cycles asynchronously — although hairs within each skin domain cycle in synchronized waves. Thus, the combinatorial defects in hair cycling and differentiation, together with concealed skin domains, account for the cyclic alopecia phenotype.

Developmental timing of hair follicle and dorsal skin innervation in mice

The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene‐related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine‐IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7– P17). Last, tyrosine hydroxylase‐IR and neuropeptide Y‐IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis. J. Comp. Neurol. 448:28–52, 2002.

Clusters of Perifollicular Macrophages in Normal Murine Skin: Physiological Degeneration of Selected Hair Follicles by Programmed Organ Deletion

In back skin sections from adolescent C57BL/6 mice, regularly distributed, perifollicular inflammatory cell clusters (PICC) were found located around the distal noncycling portion of about 2% of all hair follicles examined. The PICC and the affected hair follicles were characterized during spontaneously developed or induced hair cycle stages, using antibodies against MHC Class II,F4/80, ER-MP23, NLDC 145, CD4, CD8, γδTCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas γδTCR+ and CD8+ cells were absent. During anagen and catagen, some of the PICC+ hair follicles showed variable degenerative phenomena reminiscent of scarring alopecia: thickened basement membrane, ectopic MHC II expression, MAC infiltration into the follicle epithelium, and signs of keratinocyte apoptosis. Loss of distal outer root sheath keratinocytes was detected in 10% of PICC+ hair follicles (0.2% of all hair follicles). Because PICC were located in the vicinity of the bulge region, MAC-dependent damage to follicle stem cells might eventually lead to follicle degeneration. These perifollicular MAC clusters around selected hair follicles may indicate the existence of a physiological program of MAC-dependent controlled follicle degeneration by which damaged or malfunctioning follicles are removed by programmed organ deletion (POD).

E- and P-cadherin expression during murine hair follicle morphogenesis and cycling

The role of adhesion molecules in the control of hair follicle (HF) morphogenesis, regression and cycling is still rather enigmatic. Since the adhesion molecules E‐ and P‐cadherin (Ecad and Pcad) are functionally important, e.g. during embryonic pattern formation, we have studied their expression patterns during neonatal HF morphogenesis and cycling in C57/BL6 mice by immunohistology and semi‐quantitative RT‐PCR. The expression of both cadherins was strikingly hair cycle‐dependent and restricted to distinct anatomical HF compartments. During HF morphogenesis, hair bud keratinocytes displayed strong Ecad and Pcad immunoreactivity (IR). While neonatal epidermis showed Ecad IR in all epidermal layers, Pcad IR was restricted to the basal layer. During later stages of HF morphogenesis and during anagen IV‐VI of the adolescent murine hair cycle, the outer root sheath showed strong E‐ and Pcad IR. Instead, the outermost portion of the hair matrix and the inner root sheath displayed isolated Ecad IR, while the innermost portion of the hair matrix exhibited isolated Pcad IR. During telogen, all epidermal and follicular keratinocytes showed strong Ecad IR. This is in contrast to Pcad, whose IR was stringently restricted to matrix and secondary hair germ keratinocytes which are in closest proximity to the dermal papilla. These findings suggest that isolated or combined E‐ and/or Pcad expression is involved in follicular pattern formation by segregating HF keratinocytes into functionally distinct subpopulations; most notably, isolated Pcad expression may segregate those hair matrix keratinocytes into one functional epithelial tissue unit, which is particularly susceptible to growth control by dermal papilla‐derived morphogens. The next challenge is to define which secreted agents implicated in hair growth control modulate these follicular cadherin expression patterns, and to define how these basic parameters of HF topobiology are altered during common hair growth disorders.

Distinct Patterns of NCAM Expression Are Associated with Defined Stages of Murine Hair Follicle Morphogenesis and Regression

Hair follicle development, growth (anagen), and regression (catagen) largely result from bidirectional epithelial-mesenchymal interactions whose molecular basis is still unclear. Because adhesion molecules are critically involved in pattern formation and because the fundamental importance of neural cell adhesion molecule (NCAM) for feather development has been demonstrated, we studied the protein expression patterns of NCAM during hair follicle development and regression in the C57BL/6 mouse model. During murine hair follicle development, NCAM immunoreactivity (IR) was first detected on epithelial hair placodes and later on selected keratinocytes in the distal outer root sheath. Mesenchymal NCAM immunoreactivity (IR) was noted on fibroblasts of the future dermal papilla (DP) and the perifollicular connective tissue sheath. Fetal hair follicle elongation coincided with strong, ubiquitous dermal NCAM IR, which remained strong until the follicles entered into their first neonatal catagen. At this time, the strong interfollicular dermal NCAM IR decreased substantially. During consecutive hair cycles, mesenchymal NCAM IR was seen exclusively on DP and perifollicular connective tissue sheath fibroblasts and on the trailing cells of regressing catagen hair follicles. These highly restricted and developmentally controlled expression patterns suggest an important role for NCAM in hair follicle topobiology during morphogenesis and cyclic remodeling of this miniorgan.

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.

Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer

Abstract:  Minoxidil induces new hair growth in approximately one‐third of patients with androgenetic alopecia after 1 year of treatment. With several conflicting reports in the literature based on small‐scale studies, the current study aimed to clarify whether organ culture of human scalp anagen VI hair follicles is a suitable in vitro test system for reproducing, and experimentally dissecting, the recognized in vivo hair‐growth‐promoting capacity of minoxidil. Hair shaft elongation was studied in terminal anagen VI hair follicles microdissected from the occipital scalp of 36 healthy adults. A total of 2300 hair follicles, approximately 65 per individual, were tested using modifications of a basic organ culture protocol. It is shown here that minoxidil does not significantly increase hair shaft elongation or the duration of anagen VI in ex vivo culture despite several enhancements on the conventional methodology. This disparity to what is seen clinically in minoxidil responders may be explained by the following: (i) use of occipital (rather than frontotemporal or vertex) hair follicles; (ii) use of, already maximally growing, anagen VI hair follicles; (iii) a predominance of hair follicles from minoxidil unresponsive‐donors; (iv) use of minoxidil rather than its sulfate metabolite; and/or (v) use of a suboptimal minoxidil dosage. This disparity questions the usefulness of standard human hair follicle organ culture in minoxidil research. Unexpectedly, minoxidil even inhibited hair shaft elongation in the absence of insulin, which may indicate that the actual hair‐growth‐modulatory effects of minoxidil depend on the concomitant local presence/absence of other growth modulators.

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.

Adrenomedullin: expression and possible role in human skin and hair growth

Background  Adrenomedullin (AM) is a regulatory peptide that is synthesized and secreted by a wide number of cells and tissues. AM is a potent vasodilator, but also exerts other functions, such as regulating cell growth and antimicrobial defence. Two receptors, L1 and calcitonin receptor‐like receptor (CRLR), which are able to bind AM, have been cloned and characterized.