ARTICLE |
Correspondence to: Ralf Paus, Humboldt-Universität, Charité, Dept. of Dermatology, D-10117 Berlin, Germany.
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Summary |
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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, TCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas
TCR+ 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). (J Histochem Cytochem 46:361370, 1998)
Key Words: hair cycle, inflammation, alopecia, macrophage, lymphocyte, MHC Class II, ICAM-1, apoptosis
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Introduction |
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Inflammatory cells, such as macrophages (MAC), lymphocytes, and mast cells are not distributed entirely regularly in skin but tend to preferentially localize in certain skin regions such as the perivascular, perifollicular, or perineural dermis. In routine dermatohistological diagnosis, it is not always easy to define with any certainty whether or not such preferential cell accumulations are still physiological or already reflect pathological phenomena (
Here we describe a striking HF-related phenomenon that casts further doubt on exactly when cutaneous inflammatory cell infiltrates have to be considered pathological. In the course of analyzing hair cycle-dependent changes in the immune status of murine skin (
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Materials and Methods |
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Animals and Tissue Collection
Syngeneic female C57BL/6 mice and Balb/c mice (Charles River; Sulzfeld, Germany) were housed in community cages with 12-hr light periods at the Animal Facilities of the Biomedical Research Center, Charité, Berlin, and were fed water and mouse chow ad libitum. Skin harvesting for cryosections, embedding, and sectioning were performed as previously described (
To obtain different spontaneously developed hair cycle stages, we took back skin of mice with all HF in telogen (6- to 8-week-old animals) or in various stages of the anagen VI/catagen transition of the hair cycle (1215-week-old animals). In addition, one group consisted of mice with back skin in spontaneously developed catagen after anagen induction by depilation of 7-week-old mice (
Immunohistology
The applied primary antibodies, source, dilution, and host species are given in Table 1. Cryosections (810 µm) were incubated with avidin- and biotin-blocking solution (Vector; Burlingame, CA) if the ABC method was applied, followed by another block using either protein blocking agent (Immunotech; Krefeld, Germany) or 4% normal goat serum in TBS (Tris-buffered saline) for 20 min at room temperature (RT). The primary antibody was diluted in TBS containing 1% normal goat serum (for titers see Table 1) and applied for 1 hr (monoclonal antibodies) or overnight (polyclonal rabbit antisera). As secondary antibodies, we used biotinylated goat-anti-rat or goat-anti-hamster (Vector) diluted 1:200 in TBS containing 4% mouse normal serum for 45 min at RT, followed by an incubation with avidinbiotinalkaline phosphatase complex (Vector) 1:100 in TBS for 30 min. After washing, a routine staining procedure for alkaline phosphatase was used and sections were counterstained with Mayer's hematoxylin. The polyclonal rabbit antisera were detected using a rhodamine-coupled goat anti-rabbit secondary antibody applied for 1 hr at 37C, followed by washing and covering of sections with Vecta-Shield cover medium (Vector).
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Double immunostaining on the same section, using anti-MHC Class II antibody and anti-NLDC145 or anti-MHC Class II antibody and anti-ICAM-1, was performed as previously described (
Histochemical Stains
Various histochemical protocols were applied, following the procedures described by
Analyses
Quantitative analyses were performed on both longitudinal and horizontal sections. Comparisons among different cell types as well as co-localization studies within the same cluster of labeled cells were done on serial cross-sections using a Zeiss microscope with a camera lucida system. All HF and the various immunohistological labelings were drawn by hand on separate transparencies, superimposed and correlated thereafter.
Statistical assessment was done using the KruskallWallis test for analyses of independent groups and the MannWhitney U-test for comparison of independent pairs (
Photomicrographs were taken with a video camera and digital image analysis was performed using the computer software ISIS (Metasystems: Altlussheim, Germany).
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Results |
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In longitudinal and horizontal sections of back skin from apparently healthy adolescent female C57BL/6 mice, we confirmed the presence of regularly spaced PICC of macrophage-like cells around individual HF. These PICC as well as the affected HF were characterized during different hair cycle stages that had either developed spontaneously or had been induced by IP cyclosporin A or by depilation. Skin sections were stained with antibodies against MHC Class II, F4/80, ER-MP23, NLDC 145, CD4, CD8, TCR, IL-1R, and ICAM-1. Table 2 summarizes the key results of these immunohistological analyses, which are described in detail below.
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Normal Murine Skin Displays Perifollicular Inflammatory Cell Clusters
Macroscopically uninflamed, normal back skin of C57BL/6 mice with all HF in telogen that had not been manipulated in any way exhibited an increased density of MHC II-IR, dermal cells clustered around individual telogen HF, more or less evenly distributed throughout the back skin (Figure 1A). Such PICC were usually located in the vicinity of the distal HF epithelium, i.e., around the noncycling portion of the HF. PICC could also be seen around HF in the growth stage (anagen VI, Figure 1B) and in the regression stage of the hair cycle (catagen VII, Figure 1C) and were visible in both longitudinal and cross-sections of C57BL/6 back skin HF. Consecutive longitudinal sections showed that PICC enwrap the entire HF (Figure 1E and Figure 1F). Interestingly, PICC were also detected in the skin of cyclosporin A-treated and thus immunosuppressed animals (not shown).
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These PICC did not appear to be a strain-specific feature of C57BL/6 mice because they were also present in Balb/c mice (Figure 1D). There were no overt signs of bacterial, fungal, or parasitic infections, either macroscopically or in any of the analyzed skin sections as checked by standard histochemical stains (PAS, Gram, Giemsa, hematoxylin/eosin; not shown).
A "cartography" of PICC distribution over the entire back skin of one untreated mouse with all HF in telogen revealed a fairly homogeneous distribution of PICC+ HF (Figure 2), with a slightly (but not significantly) lower density in the lower back region compared to the neck or central back regions (p>0.05). To further analyze the distribution of PICC as well as to perform multiple labeling studies on the same cluster of cells and on the same follicle, serial horizontal cross-sections were further investigated (e.g., Figure 1D and Figure 1H). Counting the number of PICC in horizontal cross-sections of eight different mice, we found 111 of 6340 HF to be surrounded by PICC (1.8%).
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Both Pelage and Tylotrich Hair Follicles Are Associated with PICC
Among the pelage follicles of murine back skin, a subpopulation of larger, so-called tylotrich hair follicles (TF) stand out (
PICC Consist Predominantly of Macrophage-like Cells
A variety of immunohistological stains on serial horizontal sections were performed to clarify the cellular composition of PICC (Figure 3). Up to eight different stains were performed in one series of consecutive sections. To identify the same HF in follow-up sections, the camera lucida system was used, and follicles and labeling results were drawn on separate transparencies, which were then superimposed (see Materials and Methods).
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Most of the cells within a PICC were MHC II-IR (Figure 3A, Figure 3F, Figure 3I, and Figure 3K) and F4/80-IR (not shown), suggesting these cells to be macrophages (MAC) or Langerhans cells. To distinguish these two cell populations, double labeling with anti-MHC II and anti-NLDC145 antibodies was performed. Quantitative analyses of the number of cells within and outside of PICC revealed that the vast majority of cells in a PICC were not Langerhans cells (i.e., were NLDC145-negative) and that only the number of MHC II+/NLDC145- cells was significantly higher in the PICC compared to the interfollicular dermis (Figure 4).
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Only very few PICC cells exhibited weak CD4-IR, but the number of CD4+ PICC cells was higher than in the surrounding dermis (Table 2) (e.g., Figure 3N compared to Figure 3K). The number of CD8-IR cells within the PICC was not increased in comparison to other skin areas (notably, the total number of CD8-IR cells in normal C57BL/6 mouse skin is normally very low). When sections were stained with an antibody against the T-cell receptor or against CD3, no clustered perifollicular labeled cells could be detected. Because MAC can also express CD4 and because PICC display a dendritic phenotype, PICC are most likely composed of activated MAC. No obvious alterations in the number or appearance of mast cells was found at the site of PICC when histochemical stainings were used (not shown) (see Table 2).
ICAM-1-IR was upregulated both inside the PICC and on ORS keratinocytes of PICC+ HF. Of a total of 1728 analyzed HF, of which 19 had a PICC, all of these PICC except one expressed strong ICAM-1-IR (not shown).
Double labeling of ICAM-1 and MHC II on the same cross-sections of telogen back skin of five different mice, estimation of the ICAM-1-IR staining intensity (arbitrary units) of follicular keratinocytes, and measuring of the size of the associated PICC revealed that there was a statistically significant correlation between the ICAM-1-IR intensity of the ORS keratinocytes in these PICC+ follicles and the size of the PICC (Figure 5A) (r2 = 0.86 for a linear correlation; p<0.01). In addition, the probability of PICC to occur appeared to correlate with the follicular ICAM-1-IR intensity (Figure 5B).
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Some Hair Follicles with PICC Exhibit Signs of Degeneration
Analyzing mouse back skin with all HF in late anagen VI or catagen, we noted that some follicles with a large PICC showed signs of follicle degeneration. These PICC+ follicles differed in their degree of degeneration, as evidenced by the relative integrity of the basement membrane, the presence of MHC II-IR cells within the follicle epithelium, and the loss of ORS keratinocytes. The latter express strong IL-1 receptor Type I-IR (
Other HF with a large, dense, and strongly MHC II-IR PICC also showed intrafollicular MHC II-IR cells, which may derive from an upregulation of MHC Class II on follicular keratinocytes and/or from MAC penetrating the HF (Figure 3F). The ORS keratinocytes shown in the HF of Figure 3F and Figure 3G still express IL-1RI-IR (Figure 3G). The basement membrane is present, although it appears to be partially disrupted (Figure 3H). In these cases, the proximaldistal expansion of the PICC can span almost the entire HF (not shown).
The clearest evidence for follicle degeneration in macroscopically completely normalappearing anagen back skin can be seen in the follicle depicted in Figure 3KO. This series of cross-sections shows an HF with a large MHC II-IR PICC (Figure 3K). The follicle had lost all its ORS keratinocytes in this area, as judged by the absence of IL-1RI-IR (Figure 3L, arrow), and the basement membrane had largely disappeared (Figure 3M). A hematoxylin/eosin stain of the subsequent section revealed light microscopic signs of apoptosis (apoptotic bodies) close to the hair shaft, suggesting that the ORS keratinocytes had undergone apoptosis in this region (Figure 3O). This was confirmed in another example by in situ end-labeling, using a fluorescent TUNEL/Hoechst 33342 staining technique (
When eight mice of different hair cycle stages were analyzed using camera lucida-aided drawings of 6340 HF, we found this most severe type of HF degeneration in only eight HF, all of which were either in late anagen or in catagen, irrespective of whether anagen had been induced by depilation or had developed spontaneously. Approximately 10% of all PICC+ anagen and catagen HF showed signs of HF degeneration.
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Discussion |
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Here we describe for the first time that macroscopically uninflamed, normal murine back skin contains regularly distributed PICC of largely MAC-like cells. These PICC are located around the distal noncycling portion of individual, fairly regularly spaced HF. These were present even in totally unmanipulated back skin with all HF in spontaneous telogen and in the absence of overt macroscopic or histochemical signs of skin infection, thus strongly suggesting that they are a physiological phenomenon. This did not appear to be a strain-specific curiosity or a phenomenon that depends on HF pigmentation. Two immunologically quite distinct mouse strains (C57BL/6, Balb/c), one black, the other white, both exhibit PICC+ follicles. Given the relative frequency of PICC+ follicles in normal mouse skin (around 2% of all HF) and the extensive scrutiny murine skin has been subjected to in countless immunohistological analyses over more than two decades (
The presence, nature, and potential biological significance of PICC are interesting in a number of respects. They underscore the fact that mammalian skin can display substantial inflammatory cell infiltrates without any macroscopically visible signs of inflammation or the presence of infection, and that normal HF can display a large, dense infiltrate without suffering apparent damage (the vast majority of PICC+ follicles were morphologically indistinguishable from PICC- follicles, and signs of follicular degeneration were seen only in a minority of anagen VI and catagen follicles and not in telogen or early anagen follicles). A common problem in dermatopathological diagnosis is how to define exactly whether or not inflammatory cells located in the vicinity of an HF should be considered pathologically increased in number (
PICC are always located at about the level of and in close proximity to the bulge region, the seat of follicular epithelial stem cells (
Intriguingly, about 10% of the PICC+ anagen VI or catagen HF show signs of degeneration reminiscent of scarring or of "permanent" nonscarring alopecia (cf.
That PICC-associated follicle degeneration occurs only around individual anagen VI and catagen HF, i.e., follicles that are about to involute or are already in the process of regression, suggests that we are witnessing here an exaggerated form of the phagocytic activities MAC normally can display during HF regression (
Naturally, the question arises as to whether this degeneration represents a physiological program of controlled follicle deconstruction or whether this kind of follicle degeneration is essentially pathological. We currently interpret PICC-associated follicle degeneration in otherwise perfectly normal murine skin as a basically physiological mechanism for removing damaged or malfunctioning follicles by programmed organ deletion (POD). Indeed, POD is a commonly employed, entirely physiological tool for tissue remodeling, which is seen, for example, in many insect and lower vertebrate species (cf. deletion of gills and tail during frog metamorphosis) as well as during embryonal and perinatal development in all mammals including humans [e.g., involution of aortic arches and omphaloenteric duct (
Various forms of clinically perceptible, permanent alopecia [including, e.g., rare cases of androgenetic alopecia, alopecia areata, and psoriatic alopecia associated with permanent HF loss (
A critically important question to be answered by future studies is how MAC become attracted to accumulate around individual HF. One reasonable explanation is that the follicle epithelium secretes chemoattractant cytokines, particularly those that it can express constitutively, such as TNF- and IL-1
(
Which mechanisms can be envisioned that might explain any such changes in the local cytokine milieu in and around the follicle bulge and the isthmus area? Hair follicle cycling as such, which is associated with profound alterations in the cutaneous and follicular cytokine milieu (cf.
Alternatively, those follicles that have suffered some form of damage or malfunction might secrete cyto-kines that attract MAC to assist in damage repair or to initiate POD, once damage or malfunctioning has surpassed a critical threshold. Interestingly, even cyclo-sporin A-treated mice display PICC+ HF, suggesting that the signal for PICC accumulation is not cyclo-sporin A-sensitive.
In summary, our observations in murine skin suggest that the presence of even very dense perifollicular inflammatory cell infiltrates (PICC) does not necessarily indicate the presence of pathological processes. In addition, these infiltrates may serve as markers for follicles selected for programmed organ deletion (POD) and/or for HF that differ from their morphologically otherwise indistinguishable neighboring follicles in their local cytokine milieu.
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Acknowledgments |
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Supported by grants from the Deutsche Forschungsgemeinschaft to RP (Pa 345/3-2, 5-1) and to IM (Mo 644/2-1).
The continued support of Prof W. Sterry is gratefully acknowledged. We thank R. Pliet, E. Hagen, and R. Böhmer for excellent technical assistance.
Received for publication April 14, 1997; accepted September 1, 1997.
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