Generation and Cyclic Remodeling of the Hair Follicle Immune System in Mice
1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.1998.00243.x
ISSN1523-1747
AutoresRalf Paus, Carina van der Veen, Stefan B. Eichmüller, Tobias Kopp, Evelin Hagen, Sven Müller‐Röver, Udo Hofmann,
Tópico(s)Dermatology and Skin Diseases
ResumoIn this immunohistomorphometric study, we have defined basic characteristics of the hair follicle (HF) immune system during follicle morphogenesis and cycling in C57BL/6 mice, in relation to the skin immune system. Langerhans cells and γδ T cell receptor immunoreactive lymphocytes were the predominant intraepithelial hematopoietic cells in neonatal mouse skin. After their numeric increase in the epidermis, these cells migrated into the HF, although only when follicle morphogenesis was almost completed. In contrast to Langerhans cells, γδ T cell receptor immunoreactive lymphocytes entered the HF only via the epidermis. Throughout HF morphogenesis and cycling, both cell types remained strikingly restricted to the distal outer root sheath. On extremely rare occasions, CD4+ or CD8+αβTC were detected within the HF epithelium or the sebaceous gland. Major histocompatibility complex class II+, MAC-1+ cells of macrophage phenotype and numerous mast cells appeared very early on during HF development in the perifollicular dermis, and the percentage of degranulated mast cells significantly increased during the initiation of synchronized HF cycling (first catagen). During both depilation- and cyclosporine A-induced HF cycling, the numbers of intrafollicular Langerhans cells, γδ T cell receptor immunoreactive lymphocytes, and perifollicular dermal macrophages fluctuated significantly. Yet, no numeric increase of perifollicular macrophages was detectable during HF regression, questioning their proposed role in catagen induction. In summary, the HF immune system is generated fairly late during follicle development, shows striking differences to the extrafollicular skin immune system, and undergoes substantial hair cycle-associated remodeling. In addition, synchronized HF cycling is accompanied by profound alterations of the skin immune system. In this immunohistomorphometric study, we have defined basic characteristics of the hair follicle (HF) immune system during follicle morphogenesis and cycling in C57BL/6 mice, in relation to the skin immune system. Langerhans cells and γδ T cell receptor immunoreactive lymphocytes were the predominant intraepithelial hematopoietic cells in neonatal mouse skin. After their numeric increase in the epidermis, these cells migrated into the HF, although only when follicle morphogenesis was almost completed. In contrast to Langerhans cells, γδ T cell receptor immunoreactive lymphocytes entered the HF only via the epidermis. Throughout HF morphogenesis and cycling, both cell types remained strikingly restricted to the distal outer root sheath. On extremely rare occasions, CD4+ or CD8+αβTC were detected within the HF epithelium or the sebaceous gland. Major histocompatibility complex class II+, MAC-1+ cells of macrophage phenotype and numerous mast cells appeared very early on during HF development in the perifollicular dermis, and the percentage of degranulated mast cells significantly increased during the initiation of synchronized HF cycling (first catagen). During both depilation- and cyclosporine A-induced HF cycling, the numbers of intrafollicular Langerhans cells, γδ T cell receptor immunoreactive lymphocytes, and perifollicular dermal macrophages fluctuated significantly. Yet, no numeric increase of perifollicular macrophages was detectable during HF regression, questioning their proposed role in catagen induction. In summary, the HF immune system is generated fairly late during follicle development, shows striking differences to the extrafollicular skin immune system, and undergoes substantial hair cycle-associated remodeling. In addition, synchronized HF cycling is accompanied by profound alterations of the skin immune system. cyclosporine-induced hair cycle depilation-induced hair cycle T cell receptor-positive lymphocytes γδ T-cell receptor immunoreactive hair follicle(s) hair follicle immune system hmacrophages CD11b (C3 bi receptor, = macrophage marker) mast cells microscopic field post-depilation post-partum skin immune system The hair follicle (HF) displays several intriguing immunologic features. For example, in striking contrast to the epidermis with its homogeneous distribution of intraepithelial lymphocytes, dendritic epidermal T cells (DETC) within murine HF epithelium are stringently restricted to a defined sector of the distal outer root sheath (ORS) (Paus et al., 1994aPaus R. Hofmann U. Eichmüller S. Czarnetzki B.M. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.Br J Dermatol. 1994 a; 130: 281-289Crossref PubMed Scopus (78) Google Scholar). Furthermore, the inner root sheath (IRS) and hair matrix of anagen follicles do not express major histocompatibility complex (MHC) class Ia molecules (Harrist et al., 1983Harrist T.J. Ruiter D.J. Mihm Jr, M.C. Bhan A.K. Distribution of major histocompatibility antigens in normal skin.Br J Dermatol. 1983; 109: 623-633Crossref PubMed Scopus (59) Google Scholar;Westgate et al., 1991Westgate G.E. Craggs R.I. Gibson W.T. Immune privilege in hair growth.J Invest Dermatol. 1991; 97: 417-420Abstract Full Text PDF PubMed Google Scholar;Paus et al., 1994bPaus R. Eichmüller S. Hofmann U. Czarnetzki B.M. Expression of classical and nonclassical MHC class I antigens in murine hair follicles.Br J Dermatol. 1994 b; 131: 177-183Crossref PubMed Scopus (75) Google Scholar) so that the proximal anagen follicle epithelium may enjoy some form of “immune privilege” (Billingham and Silvers, 1971Billingham R.E. Silvers W.K. A biologists reflection on dermatology.J Invest Dermatol. 1971; 57: 495-499Google Scholar;Westgate et al., 1991Westgate G.E. Craggs R.I. Gibson W.T. Immune privilege in hair growth.J Invest Dermatol. 1991; 97: 417-420Abstract Full Text PDF PubMed Google Scholar;Paus et al., 1994bPaus R. Eichmüller S. Hofmann U. Czarnetzki B.M. Expression of classical and nonclassical MHC class I antigens in murine hair follicles.Br J Dermatol. 1994 b; 131: 177-183Crossref PubMed Scopus (75) Google Scholar,Paus et al., 1994cPaus R. Slominski A. Czarnetzki B.M. Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I-expression in the anagen hair bulb?.Yale J Biol Med. 1994 c; 66: 541-554Google Scholar;Paus, 1997Paus R. Immunology of the hair follicle.The Skin Immune System. In: Bos JD (ed.). CRC Press, Boca Raton1997: 377-398Google Scholar). In addition, the distal ORS expresses so-called nonclassical MHC class Ib molecules (Paus et al., 1994bPaus R. Eichmüller S. Hofmann U. Czarnetzki B.M. Expression of classical and nonclassical MHC class I antigens in murine hair follicles.Br J Dermatol. 1994 b; 131: 177-183Crossref PubMed Scopus (75) Google Scholar;Rückert et al., 1998Rückert R. Hofmann U. van der Veen C. Bulfone-paus S. Paus R. MHC class I expression in murine skin: developmentally controlled and strikingly restricted intraepithelial expression during hair follicle morphogenesis and cycling, and response to cytokine treatment in vivo.J Invest Dermatol. 1998; 111: 25-30Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Taken together, this has raised the possibility that the HF has a distinct “hair follicle immune system” (HIS) (Paus, 1997Paus R. Immunology of the hair follicle.The Skin Immune System. In: Bos JD (ed.). CRC Press, Boca Raton1997: 377-398Google Scholar) that differs from the surrounding “skin immune system” (SIS) (Bos, 1997Bos J.D. The Skin Immune System. (ed.). CRC Press, Boca Raton1997Google Scholar). Synchronized HF cycling is also associated with substantial alterations of the skin immune status and of standard skin immune responses (Claesson and Hardt, 1970Claesson M.H. Hardt F. The influence of the hair follicle phase on the survival time of skin allografts in the mouse.Transplantation. 1970; 10: 349-351Crossref PubMed Scopus (6) Google Scholar;Westgate et al., 1991Westgate G.E. Craggs R.I. Gibson W.T. Immune privilege in hair growth.J Invest Dermatol. 1991; 97: 417-420Abstract Full Text PDF PubMed Google Scholar;Paus et al., 1994bPaus R. Eichmüller S. Hofmann U. Czarnetzki B.M. Expression of classical and nonclassical MHC class I antigens in murine hair follicles.Br J Dermatol. 1994 b; 131: 177-183Crossref PubMed Scopus (75) Google Scholar;Hofmann et al., 1996Hofmann U. Tokura Y. Nishijima T. Takigawa M. Paus R. Hair cycle dependent changes in skin immune functions: Anagen associated depression of sensitization for contact hypersensitivity in mice.J Invest Dermatol. 1996; 106: 598-604Crossref PubMed Scopus (38) Google Scholar,Hofmann et al., 1998Hofmann U. Tokura Y. Rückert R. Paus R. The anagen hair cycle induces systemic immunosuppression of contact hypersensitivity in mice.Cellular Immunology. in press. 1998Crossref Scopus (19) Google Scholar;Tokura et al., 1997Tokura Y. Hofmann U. Müller-röver S. et al.Spontaneous hair follicle cycling may influence the development of murine contact photosensitivity by modulating keratinocyte cytokine production.Cell Immunol. 1997; 178: 172-179Crossref PubMed Scopus (15) Google Scholar). In turn, the dermal SIS, namely mast cells (MC) and macrophages (MAC), may be involved in hair growth control (Parakkal, 1969Parakkal P.F. Role of macrophages in collagen resorption during hair growth cycle.J Ultrastruct Res. 1969; 29: 210-217Crossref PubMed Scopus (82) Google Scholar;Westgate et al., 1991Westgate G.E. Craggs R.I. Gibson W.T. Immune privilege in hair growth.J Invest Dermatol. 1991; 97: 417-420Abstract Full Text PDF PubMed Google Scholar;Paus et al., 1994dPaus R. Maurer M. Slominski A. Czarnetzki B.M. Mast cell involvement in murine hair growth.Dev Biol. 1994 d; 163: 230-240Crossref PubMed Scopus (118) Google Scholar;Botchkarev et al., 1995Botchkarev V.A. Paus R. Czarnetzki B.M. Kupriyanov V.S. Gordon D.S. Johansson O. Hair cycle-dependent changes in mast cell histochemistry in murine skin.Arch Dermatol Res. 1995; 287: 683-686Crossref PubMed Scopus (22) Google Scholar,Botchkarev et al., 1997Botchkarev V.A. Eichmüller S. Peters E.M.J. Pietsch P. Johansson O. Maurer M. Paus R. A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle-dependent changes in mast cell-nerve fiber contacts in murine skin.Arch Dermatol Res. 1997; 289: 292-302Crossref PubMed Scopus (100) Google Scholar;Maurer et al., 1995Maurer M. Paus R. Czarnetzki B.M. Mast cells as modulators of hair follicle cycling.Exp Dermatol. 1995; 4: 266-271Crossref PubMed Scopus (55) Google Scholar,Maurer et al., 1997Maurer M. Fischer E. Handjiski B. von Stebut E. Algermissen B. Bavandi A. Paus R. Activated skin mast cells are involved in murine hair follicle regression.Lab Invest. 1997; 77: 319-332PubMed Google Scholar). Yet, even basic data on HF immunology are still missing. In particular, the following issues are still waiting to be clarified: (i) the migration of hematopoietic cells into the HF and the stepwise installation of the HIS during follicle morphogenesis, (ii) the exact distribution of Langerhans cells and αβ T cells throughout the entire HF epithelium, (iii) differences in the organization of the HIS during the hair cycle, and (iv) hair cycle-dependent changes in the number and distribution of extrafollicular constituents of the SIS, specifically of MAC. To clarify these issues, we have studied the assembly of the HIS during neonatal HF morphogenesis in mice by standardized, quantitative immunohistomorphometry, using antibodies that demarcate γδ T cells, αβ T cells (CD4, CD8), Langerhans cells, MAC, and MC. With the exception of MC, which have already been extensively characterized (Paus et al., 1994dPaus R. Maurer M. Slominski A. Czarnetzki B.M. Mast cell involvement in murine hair growth.Dev Biol. 1994 d; 163: 230-240Crossref PubMed Scopus (118) Google Scholar;Botchkarev et al., 1995Botchkarev V.A. Paus R. Czarnetzki B.M. Kupriyanov V.S. Gordon D.S. Johansson O. Hair cycle-dependent changes in mast cell histochemistry in murine skin.Arch Dermatol Res. 1995; 287: 683-686Crossref PubMed Scopus (22) Google Scholar,Botchkarev et al., 1997Botchkarev V.A. Eichmüller S. Peters E.M.J. Pietsch P. Johansson O. Maurer M. Paus R. A simple immunofluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle-dependent changes in mast cell-nerve fiber contacts in murine skin.Arch Dermatol Res. 1997; 289: 292-302Crossref PubMed Scopus (100) Google Scholar;Maurer et al., 1997Maurer M. Fischer E. Handjiski B. von Stebut E. Algermissen B. Bavandi A. Paus R. Activated skin mast cells are involved in murine hair follicle regression.Lab Invest. 1997; 77: 319-332PubMed Google Scholar), the distribution and number of these immunocytes were also assessed during all stages of the hair cycle in adolescent mice, comparing pharmacologically and depilation-induced HF cycling. Tissue banks were prepared from neonatal and adolescent back skin of C57BL/6 mice obtained from Charles River (Sulzfeld, Germany) as described in detail (Paus et al., 1994ePaus R. Handjiski B. Czarnetzki B.M. Eichmüller S. A murine model for inducing and manipulating hair follicle regression (catagen): effects of dexamethasone and cyclosporin A.J Invest Dermatol. 1994 e; 103: 143-147Crossref PubMed Scopus (111) Google Scholar,Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-paus S. Eichmüller S. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Anagen was induced by depilation of hair shafts on the back of mice with all follicles in telogen, as described (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (208) Google Scholar,Paus et al., 1994fPaus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice. Induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994 f; 144: 719-734PubMed Google Scholar). In order to be able to distinguish changes in HF and skin immune parameters related to the trauma of depilation, which induces a discrete wound healing response early on during anagen development (Argyris, 1967Argyris T.S. Hair growth induced by damage.Adv Biol Skin. 1967; 9: 339-345Google Scholar), from truly hair cycling-related changes, a second, pharmacologic method of anagen induction was employed by i.p. application of cyclosporine A (CsA, Sandimmun; Sandoz, Basel, Switzerland). CsA was given once per day at a dosage of 250 mg per kg on days 0, 1, and 3, which initiates a new synchronized hair cycle (cyclosporine-induced hair cycle, CsA-HC) (Paus et al., 1989Paus R. Stenn K.S. Link R.E. The induction of anagen hair growth in telogen mouse skin by cyclosporine A administration.Lab Invest. 1989; 60: 365-369PubMed Google Scholar,Paus et al., 1996aPaus R. Böttge J.A. Henz B.M. Maurer M. Hair growth control by immunosuppression.Arch Dermatol Res. 1996 a; 288: 408-410Crossref PubMed Scopus (33) Google Scholar). In both depilation- and CsA-induced anagen HF, catagen develops spontaneously after 17–19 d (Paus et al., 1994ePaus R. Handjiski B. Czarnetzki B.M. Eichmüller S. A murine model for inducing and manipulating hair follicle regression (catagen): effects of dexamethasone and cyclosporin A.J Invest Dermatol. 1994 e; 103: 143-147Crossref PubMed Scopus (111) Google Scholar,Paus et al., 1996aPaus R. Böttge J.A. Henz B.M. Maurer M. Hair growth control by immunosuppression.Arch Dermatol Res. 1996 a; 288: 408-410Crossref PubMed Scopus (33) Google Scholar). The time course of HF cycling after anagen induction by depilation (dep-HC) and after anagen induction by CsA (CsA-HC) were compared by quantitative histomorphometry (Paus et al., 1994ePaus R. Handjiski B. Czarnetzki B.M. Eichmüller S. A murine model for inducing and manipulating hair follicle regression (catagen): effects of dexamethasone and cyclosporin A.J Invest Dermatol. 1994 e; 103: 143-147Crossref PubMed Scopus (111) Google Scholar,Paus et al., 1996bPaus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair regrowth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996 b; 56: 4438-4443PubMed Google Scholar;Maurer et al., 1997Maurer M. Fischer E. Handjiski B. von Stebut E. Algermissen B. Bavandi A. Paus R. Activated skin mast cells are involved in murine hair follicle regression.Lab Invest. 1997; 77: 319-332PubMed Google Scholar), analyzing 20 HF from each of five different mice during various time points of the dep-HC and the CsA-HC. This revealed that, in CsA-HC, anagen I was first detected 5 d after the first CsA injection, whereas anagen I could already be found at day 1 post-depilation (p.d.) in dep-HC. Thereafter the dynamics and time course of induced HF cycling were virtually identical between dep-HC and CsA-HC (data not shown). Thus, there is a time lap of 5 d between the onset of dep-HC and the onset of CsA-HC, after which both types of induced HF cycling show a practically identical anagen development. Back skin was dissected at the level of the subcutis just below the panniculus carnosus. For routine histology, paravertebral probes were taken from the upper half of the dissected skin and fixed in 5% buffered formalin, pH = 7.4. For immunohistology, skin probes from the lower half of the back skin were prepared for obtaining full longitudinal HF cryosections, using a special embedding technique (Paus et al., 1994aPaus R. Hofmann U. Eichmüller S. Czarnetzki B.M. Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.Br J Dermatol. 1994 a; 130: 281-289Crossref PubMed Scopus (78) Google Scholar,Paus et al., 1994bPaus R. Eichmüller S. Hofmann U. Czarnetzki B.M. Expression of classical and nonclassical MHC class I antigens in murine hair follicles.Br J Dermatol. 1994 b; 131: 177-183Crossref PubMed Scopus (75) Google Scholar). In order to obtain samples of all stages of HF morphogenesis, neonatal mice were sacrificed on eight different days after birth [days 1, 3, 5, 7, 9, 11, 17, and 20 post-partum (p.p.)], whereas specimens of the dep-HC and CsA-HC were obtained on days 1, 3, 5, 8, 12, 17, 19, 25, and 34 p.d., or on days 4, 6, 8, 11, 15, 21, and 31 after the first CsA injection. These murine back skin samples with their well-defined hair cycle stages were compared with completely unmanipulated adolescent mouse skin with all HF in telogen (termed day 0) from 6 to 8 wk old C57BL/6 mice. The primary antibodies used for immunohistology and their source, dilution, and host species are listed in Table 1. All incubation steps were interspersed by washing with Tris-buffered saline (TBS, 0.05 M, pH 7.6; 3 × 5 min). Non-specific binding was blocked by application of an avidin-biotin blocking kit solution (Vector Laboratories, Burlingame, VT) and by 5% bovine normal serum in TBS. Thereafter, sections were incubated with the primary antibody, diluted in TBS containing 1% bovine normal serum for 45 min (for dilution see Table 1), followed by biotinylated secondary antibodies (goat anti-rat or rabbit anti-hamster, Dianova/Pharmingen; 1:200 in TBS containing 4% normal mouse serum; 30 min). Then the ABC-AP complex was added to the slides (Vector Laboratories; 1:100; 30 min), followed by staining for alkaline phosphatase, and counterstaining in Mayer's hemalaun (Handjiski et al., 1994Handjiski B.K. Eichmüller S. Hofmann U. Czarnetzki B.M. Paus R. Alkaline phosphatase activity and localization during the murine hair cycle.Br J Dermatol. 1994; 131: 303-310Crossref PubMed Scopus (127) Google Scholar). Double-labeling studies (see Results) were performed according to a method that we had developed for labeling skin sections with two primary antibodies from the same species (Eichmüller et al., 1996Eichmüller S. Stevenson P.A. Paus R. A new method for double immunolabelling with primary antibodies from identical species.J Immunol Meth. 1996; 190: 255-265Crossref PubMed Scopus (35) Google Scholar). Negative controls were obtained by omission of primary antibody, or by using nonspecific hamster or rat IgG instead. Because the expression of all tested antigens in one or all of these organs is well documented (e.g.,Schuler, 1991Schuler G. Epidermal Langerhans Cells. (ed.). CRC Press, Boca Raton1991Google Scholar;Janeway and Travers, 1997Janeway C.A. Travers P. Immunobiology. Current Biology Ltd/Garland Publishing, London1997Google Scholar;Bos, 1997Bos J.D. The Skin Immune System. (ed.). CRC Press, Boca Raton1997Google Scholar), thymus and spleen as well as murine skin itself served as positive controls. All control staining results for the employed primary antibodies yielded the expected immunoreactivity pattern (Table 1). Giemsa staining was employed on paraffin-embedded longitudinal skin sections to identify MC by their characteristic morphology and the presence of metachromatic granules (Paus et al., 1994dPaus R. Maurer M. Slominski A. Czarnetzki B.M. Mast cell involvement in murine hair growth.Dev Biol. 1994 d; 163: 230-240Crossref PubMed Scopus (118) Google Scholar).Table IEmployed antibodies, source, and dilutionAntigenCloneMainly expressed bySourceSpeciesDilutionpan-γδTCRGL3aGoodman and Lefrancois, 1989γδ-receptor bearing T cellsiGoldsmith, 1991Pharmingenhamster1/75CD4 (L3T4)RM4-5T helper lymphocytes,iGoldsmith, 1991 MACjRoulston etal,1995;Pharmingenrat1/400CD8 (Ly2)53-6.7bLedbetter etal,1980cytotoxic/suppresser T lymphocytes'Pharmingenrat1/100NLDC145NLDC145cKraal etal,1986Langerhans cells; dendritic cellscKraal etal,1986BMArat1/250MAC-1 (CD11b)M1/70dSpringer et al, 1979MACdSpringer et al, 1979Pharmingenrat1/8000MHC IIER-TR3evan Vliet et al,1984Langerhans cells (epidermis);iGoldsmith, 1991 MAC, dermal dendritic cellskDuraiswamy et al,1994;BMArat1/200c-Kit2B8fIkuta and Weissman, 1992MC, melanocyteslHamann et al,1995Pharmingenrat1/200E-cadherinECCD-1gYoshida-Noro etal,1984keratinocytesmHirai etal,1989Zymedrat1/1500aE/β7 (CD103)m290hKilshaw and Murant, 1990intraepithelial T cellsnLefTancois etal,1994.Pharmingenrat1/250a Goodman and Lefrancois, 1989Goodman T. Lefrancois L. Intraepithelial lymphocytes. Anatomical site, not T cell receptor form, dictates phenotype and function.J Exp Med. 1989; 170: 1569-1581Crossref PubMed Scopus (323) Google Scholarb Ledbetter et al., 1980Ledbetter J.A. Rouse R.V. Micklem H.S. Herzenberg L.A. T cell subsets defined by expression of Lyt-1,2,3 and Thy-1 antigens. Two-parameter immunofluorescence and cytotoxicity analysis with monoclonal antibodies modifies current views.J Exp Med. 1980; 152: 280-295Crossref PubMed Scopus (474) Google Scholarc Kraal et al., 1986Kraal G. Breel M. Janse M. Bruin G. Langerhans cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody.J Exp Med. 1986; 163: 981-997Crossref PubMed Scopus (369) Google Scholard Springer et al., 1979Springer T. Galfre G. Secher D.S. Milstein C. Mac-1: a macrophage differentiation antigen identified by monoclonal antibody.Eur J Immunol. 1979; 9: 301-306Crossref PubMed Scopus (860) Google Scholare van Vliet et al., 1984van Vliet E. Melis M. van Ewijk W. Monoclonal antibodies to stromal cell types of the mouse thymus.Eur J Immunol. 1984; 14: 524-529Crossref PubMed Scopus (189) Google Scholarf Ikuta and Weissman, 1992Ikuta K. Weissman I.L. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.Proc Natl Acad Sci USA. 1992; 89: 1502-1506Crossref PubMed Scopus (478) Google Scholarg Yoshida-noro et al., 1984Yoshida-noro C. Suzuki N. Takeichi M. Molecular nature of the calcium-dependent cell-cell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody.Dev Biol. 1984; 101: 19-27Crossref PubMed Scopus (205) Google Scholarh Kilshaw and Murant, 1990Kilshaw P.J. Murant S.J. A new surface antigen on intraepithelial lymphocytes in the intestine.Eur J Immunol. 1990; 20: 2201-2207Crossref PubMed Scopus (177) Google Scholari Goldsmith, 1991Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University of Press, New York1991Google Scholarj Roulston etal,1995;k Duraiswamy et al,1994;l Hamann et al., 1995Hamann K. Haas N. Grabbe J. Czarnetzki B.M. Expression of stem cell factor in cutaneous mastocytosis.Br J Dermatol. 1995; 133: 203-208Crossref PubMed Scopus (63) Google Scholarm Hirai et al., 1989Hirai Y. Nose A. Kobayashi S. Takeichi M. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis.Development. 1989; 105: 271-277PubMed Google Scholarn LefTancois etal,1994. Open table in a new tab Based on functional and anatomical considerations, the epithelial and mesenchymal skin regions were divided into defined tissue compartments, as depicted in Figure 1. Twenty HF were analyzed for every mouse. Immunoreactive cells per epithelial compartment were counted after orienting a longitudinally cut HF into the center of the microscopic field [MF, ×400; strictly avoiding overlap of the interfollicular epidermal compartment (Figure 1,I)]. For evaluation of the mesenchymal compartments, a 31 μm wide strip (using an ocular micrometer grid) was virtually placed around the HF (Figure 1, compartments A, B, and C). During follicle morphogenesis, the dermal perifollicular mesenchyme (31 μm wide strip) was counted together as one “dermal compartment.” Nine different stages of follicle morphogenesis (stages 0–8) can be distinguished in neonatal skin (Vielkind et al., 1995Vielkind U. Sebzda M.K. Gibson I.R. Hardy M.H. Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8.Acta Anat. 1995; 152: 93-109Crossref PubMed Scopus (53) Google Scholar;Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-paus S. Eichmüller S. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar) (see Results). The compartment scheme for mature HF (Figure 1) was employed as soon as follicles had reached developmental stage 6 and later. For the evaluation of mast cell numbers and degranulation patterns, all Giemsa-positive cells were counted within the perifollicular dermis and subcutis, and their granulation pattern was recorded (“not degranulated,” no extracellular metachromatic granules visible;“strongly degranulated,” > six extracellular mast cell granules). To evaluate the percentage of MHC II+ cells among all dermal cells in telogen skin, and for comparing the absolute numbers of dermal MHC II/CD4 double-positive cells during telogen and anagen VI, a defined square reference area (0.015 mm2) was randomly placed into the interfollicular dermis by the ocular micrometer grid (10 MF per mouse; five mice per day). Photomicrographs were taken with the Zeiss Axiophot system (Zeiss, Oberkochen, Germany). All immunoreactivity patterns were qualitatively recorded in standardized, computer-generated schemes of murine HF morphogenesis and cycling (Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-paus S. Eichmüller S. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar) by at least two independent observers. In neonatal mouse skin, a heterogeneous mixture of different stages of HF development is found during each of the first 7–8 d of life (Vielkind et al., 1995Vielkind U. Sebzda M.K. Gibson I.R. Hardy M.H. Dynamics of Merkel cell patterns in developing hair follicles in the dorsal skin of mice, demonstrated by a monoclonal antibody to mouse keratin 8.Acta Anat. 1995; 152: 93-109Crossref PubMed Scopus (53) Google Scholar;Paus et al., 1997Paus R. Foitzik K. Welker P. Bulfone-paus S. Eichmüller S. Transforming growth factor-beta receptor type I and type II expression during murine hair follicle development and cycling.J Invest Dermatol. 1997; 109: 518-526Abstract Full Text PDF PubMed Scopus (106) Google Scholar). Because our histomorphometric results were mostly obtained with reference to defined days of postnatal skin development, the prevalence of each stage of HF morphogenesis during each of these days was determined (i.e., on days 1, 3, 5, 7 p.p.; counting a total of 100 HF per day, i.e., 20 follicles each from five different mice). From this analysis, the percentage of follicles in late stages of follicle morphogenesis (stages 7 or 8) was calculated. The mean cell numbers counted per compartment and mouse served as the basis for statistical analyses of immunocyte numbers. For each time point during neonatal follicle morphogenesis, dep-HC, or CsA-HC, and for each antigen, five mice (125 mice in total, >7500 HF) and 20 HF per mouse were analyzed. Statistical significance was estimated using the Kruskal–Wallis test for each compartment and – if positive – the Mann–Whitney U test for comparing pairs. For graphic representation, the mean and standard error of the mean as well as the p value were calculated. During the early postnatal days, HF in very different stages of their development (e.g., Figure 2d) were found located next to each other within the same skin section, representing tylotrich HF and the bulk of pelage follicles, which develop at various time points in the perinatal period (Vielkind et al., 1995Vielkind U. Sebzda M.K. Gibson I.R. Hardy M.H. Dynamics of Merke
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