Ryusuke Imai

The effect of hepatocyte growth factor/scatter factor on human hair follicle growth

The effect of hepatocyte growth factor/scatter factor (HGF/SF) on human hair follicle growth was examined using a serum-free organ culture system. The DNA synthesis in human hair follicles and elongation of the hair shaft were measured subsequent to the follicle isolation and culture at 31 degrees C in 95% O2-5% CO2 for 72 h. Results showed that HGF/SF significantly increased 3H-thymidine (P < 0.001) incorporation and hair follicle length (P < 0.05). The effect of HGF/SF was dose-dependent with a maximal stimulation at 10 ng/ml.

Organ culture of human hair follicles in serum-free medium

Human hair follicles were cultured in serum-free media at 31‡C in an atmosphere containing 95% O2 and 5% CO2. Results showed that the length of the cultured hair increased time dependently for 96 h. Histological findings revealed that the hair germinative cells maintained their normal morphology throughout the 96 h culture period. DNA synthesis in the hair bulb also increased time dependently for 96 h. Autoradiographs of 3H-thymidinelabelled follicles indicated that they were localized in the germinative cells below Aubers critical line. The effects of minoxidil sulphate on DNA synthesis in this culture system were concentration dependent. Minoxidil sulphate at concentrations of 10−10,10−9 and 10−8M significantly increased DNA synthesis compared with DNA synthesis in the control medium. Autoradiographs of the follicles cultured in 10−10M minoxidil sulphate showed that 3H-thymidine localized primarily in the germinative cells below Aubers critical line. These results suggest that this organ culture system may be useful for studying DNA synthesis by hair germinative cells in serum-free media.

Dermal papilla cells express hepatocyte growth factor

In the induction, development and maintenance of hair follicles, it is thought that an epithelial-mesenchymal interaction is important and that the dermal papilla plays some important roles. Hepatocyte growth factor is a multifunctional polypeptide which acts as mitogen, motogen or morphogen depending on the biological context. Recently, we found that HGF stimulates hair follicle growth in a mouse organ culture system, and therefore proceeded to investigate the expression of HGF on cultured human dermal papilla cells (DPC) and the effect of HGF on cultured human keratinocytes derived from hair bulb. Using an enzyme immuno assay, HGF immunoreactivities were not detected in conditioned media of DPC that were either non-treated or treated with TGF-beta, but were detected in conditioned media of DPC treated with IL1-alpha, TNF-alpha and TPA. Using the reverse transcription-polymerase chain reaction (RT-PCR) method, HGF mRNA was also detected in DPC. This expression was enhanced by IL1-alpha, TNF-alpha and TPA, but suppressed by TGF-beta. Furthermore, HGF stimulated the DNA synthesis in keratinocytes derived from human hair bulb in a dose-dependent manner. These results indicate that DPC express HGF in vitro and that HGF stimulates the growth of human keratinocytes derived from hair bulb in vitro.

Organ culture conditions of human hair follicles

Experimental results revealed that although [3H]thymidine uptake in the hair bulb increased time dependently for 12 days under normal culture conditions (95% air-5% CO2 at 37 degrees C), striking morphological changes occurred in the hair bulb cells as demonstrated by histological findings. As such, organ culture conditions applicable to human hair follicles were studied utilizing observations from both histology and DNA synthesis. We found that culture conditions of 95% O2-5% CO2 at 31 degrees C were superior when compared to normal culture conditions for cultures of human hair follicles when attempting to maintain the normal morphology of hair germinative cells. The hair bulb and the germinative cells successfully maintained their normal morphology throughout the 96 and 48 h culture period, respectively. Autoradiographs of [3H]thymidine-labeled follicles showed localization in the germinative cells below Aubers critical line. Hair bulb DNA synthesis increased time dependently for 96 h after culture initiation. Under conditions of 95% O2-5% CO2 at 31 degrees C, the synthesis of DNA in hair germinative cells was observed. Such an organ culture method may prove useful for studies on the human hair growth mechanism.

Organ culture of mouse vibrissal hair follicles in serum-free medium.

We developed a method for organ culture of mouse vibrissal hair follicles in a serum‐free medium. Cultures conducted at 31°C in 95% O2‐5% CO2 were found to be suitable for the follicles, with several findings of considerable interest pertaining to hair growth. During the 96 h culture period, the length of the isolated follicles significantly increased; the hair bulb cells maintained their normal morphology; and DNA and protein synthesis within the bulb increased time‐dependently. Furthermore, autoradiography showed that 3H‐thymidine‐labeling was localized in the matrix cells below Aubers critical line in the hair bulb; 3H‐leucine‐labeling was found in the epithelial region; and 35S‐cysteine‐labeling was detected in the cortex of hair, particularly in the keratogenous zone. These results indicate that the culture system using mouse vibrissal hair would be potentially useful as an effective model for examination of hair growth.

Increased HLA-DR+ T-lymphocyte population in peripheral blood of alopecia areata.

The populations of activated T‐cell subsets [HLA‐DR+ ‐Leu 4+ cells, interleukin 2 receptor positive (IL‐2R +)‐Leu 4+ cells] in the peripheral blood of patients with alopecia areata (AA) were investigated using double direct immunofluorescence staining. Fifty‐eight patients with AA were classified into one of three types: those with inactive single AA (type 1) lesions, active multiple alopecia areata (MAA) lesions and active alopecia totalis (AT) (type 2) and chronic alopecia universalis (AU) (type 3). Compared to normal controls, high percentages of HLA‐DR+‐Leu 4+ cells were detected in types 2 and 3 AA patients, but not in type 1 AA patients. These findings suggest that T cells are activated in the peripheral blood of active MAA, AT and chronic AU.

The effect of various cytokines on hair growth of mouse vibrissae in organ culture.

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional polypeptide which acts as a mitogen, motogen or morphogen depending on the biological context. In this study, we examined the effect of HGF on hair growth using a serum-free organ culture system. Vibrissal hair follicles isolated from newborn mice were cultured at 31 degrees C in 95% O2-5%CO2 for 72 h in the presence of various cytokines or growth factors. DNA, protein synthesis and elongation of the hair shaft in the hair follicles were measured. Among the agents tested, only HGF significantly increased hair follicle length (P < 0.001) and 3H-thymidine (P < 0.001) incorporation. The effect of HGF was dose-dependent, with maximal stimulation obtained at 10 ng/ml. The increase in hair follicle length and thymidine incorporation were specifically inhibited by a neutralizing antibody against HGF. These results indicate that HGF is able to promote hair growth and may have clinical utility in this regard.

Effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in hair bulb cells

We analysed the effects of cytokines, anti-cancer agents and cocarcinogen on DNA synthesis in human hair germinative cells cultured in serum-free media. Epidermal growth factor and gamma interferon were found to inhibit DNA synthesis slightly, while strong inhibition was demonstrated by doxorubicin, cytosine arabinoside and tetradecanoyl-phorbolacetate. Basic fibroblast growth factor had very little influence on DNA synthesis. This organ culture model in serum-free media is a useful method by which to examine the effects of various cytokines and drugs on DNA synthesis in hair germinative cells and/or to study the pathogenesis of various alopecia diseases.

Changes in populations of HLA-DR+CD3+ cells and CD57-CD16+ cells in alopecia areata after corticosteroid therapy

We investigated the populations of activated T (HLA-DR+CD3+) cells and natural killer (CD57-CD16+) cells in the peripheral blood of patients with various types of alopecia areata (AA) and noted any changes that occurred in the said populations after administration of local and systemic corticosteroid therapy. In type 2 (severe multiple AA and alopecia totalis) and type 3 (alopecia universalis), the mean percentages of HLA-DR+CD3+ cells and CD57-CD16+ cells were significantly higher when compared with those of the normal controls. The percentages of both subsets in type 1 (mild AA) and the normal controls were consistent. Twenty-four patients in types 2 and 3 had received corticosteroid treatment, and all patients experienced new hair growth. With the changes in disease activity, the populations of HLA-DR+CD3+ cells in these patients after corticosteroid therapy significantly decreased when compared with those recorded prior to treatment. Subsequent to treatment, the mean percentages of CD57-CD16+ cells decreased to levels that were not significant relative to that of the normal controls. These findings indicate that HLA-DR+CD3+ and CD57-CD16+ cells in the peripheral blood of patients with AA may be correlated with the disease activity of AA.

Analysis of T cell, activated T cell and NK cell subsets in peripheral blood lymphocytes from patients with alopecia areata

Forty-one patients with severe alopecia areata (AA) were studied. They were classified into four types according to clinical manifestations (type 1: fixed alopecia universalis or multiple AA, type 2: active alopecia totalis, type 3: active multiple AA and type 4: active multiple AA with anti-thyroid antibodies). T cell, activated T cell and NK cell subsets in the peripheral blood lymphocytes were investigated using a fluorescence activated cell sorter. (1) The percentage of Leu4 + cells in total lymphocytes showed a significant decrease in AA with types 1, 3 and 4 when compared to those of the normal controls. (2) The percentages of Leu3a+-DR+ cells in Leu3a+ cells and of Leu2a +-DR + cells in Leu2a + cells were significantly higher in AA with types 1, 3 and 4 than those of the normal controls. (3) The percentages of Leu11 + cells and Leu7 + Leu11+ cells in total lymphocytes were significantly high in AA with type 3. Leu7 − Leu11 + cells were elevated in AA with types 1 and 3. (4) The percentage increase of Leu7 − Leul l + cells was proportional to the disease activity in AA with type 3. The increase of activated T cell in peripheral blood lymphocytes from AA suggests that immune mechanisms are involved in the pathogenesis of AA. In type 3 AA, NK cell may play an important role in the pathogenesis of AA.