To help elucidate the factors responsible for the infundibular changes seen in acne, the human sebaceous pilosebaceous infundibulum was isolated by microdissection and maintained for 7 d in keratinocyte serum-free medium supplemented with 50 micrograms/ml bovine pituitary extract, 100 units/ml penicillin and streptomycin, 2.5 micrograms/ml amphotericin B and CaCl2(10H2O) to give a final Ca2+ concentration of 2 mM. Infundibular structure was maintained over 7 d in this medium; the pattern of cell division mimicked that in vivo. The rate of cell division was significantly higher than previously described for infundibula maintained in supplemented William's E medium, and moreover did not fall over 7 d. The addition of 1 ng/ml interleukin-1 alpha (IL-1 alpha) caused hypercornification of the infundibulum similar to that seen in comedones; this could be blocked by 1000 ng/ml interleukin-1 receptor antagonist (IL-1ra). In about 20% of subjects there was spontaneous hypercornification of the infundibulum that could be blocked by 1000 ng/ml IL-1ra, suggesting that the infundibulum is capable of synthesising IL-1 alpha. The addition of 5 ng/ml epidermal growth factor or 5 ng/ml transforming growth factor-alpha to the medium caused a disorganisation of the keratinocytes of the infundibulum that resulted in rupturing similar to that seen in the more severe, purulent grades of acne. The addition of 1 microM 13-cis retinoic acid caused a significant reduction in the rate of DNA synthesis and apparent parakeratosis. We are now, therefore, able to model histologically the major infundibular changes in acne.

We have previously reported that human sebaceous glands can be maintained for up to 14 d as whole organs with full retention of the physiological rate and pattern of new cell formation, but we have also reported that the newly formed cells did not differentiate normally, causing a progressive loss of lipogenesis in vitro. We now show that this abnormal sebocyte differentiation was attributable to the presence of epidermal growth factor (EGF) and phenol red in our maintenance medium. In their absence, human sebaceous glands apparently retain in vivo rates of cell division and lipogenesis over 7 d of maintenance in addition to a retention of in situ morphology. This is reversible on the re-addition of 10 ng EGF/ml and 10 mg phenol red/ml. The addition of 600 pM 17 beta-estradiol results in a significant fall in the rate of lipogenesis over 7 d of maintenance, without affecting the rate of cell division. This effect is apparently due to abnormal differentiation of newly formed sebocytes. Neither 1 nM testosterone nor 1 nM dihydrotestosterone (DHT) has any effect on rates of cell division of lipogenesis over 7 d. In the presence of phenol red, however, 1 nM testosterone or 1 nM DHT cause a significant reduction in the rate of lipogenesis over 7 d of maintenance. One micromolar 13-cis retinoic acid caused a significant reduction in the rate of lipogenesis over 7 d in both the presence and absence of phenol red. These findings show that we can model the physiological effects of steroids, EGF, and 13-cis retinoic acid in vitro.

The human sebaceous pilosebaceous infundibulum was isolated and maintained in medium for up to 7 d. Freshly isolated infundibula were found to express keratins 1, 5, 6, 16, and 17, as determined by immunohistochemistry. In addition, freshly isolated infundibula expressed filaggrin, profilaggrin, involucrin, cornifin alpha, and loricrin. This pattern of expression was retained over 7 d. The addition of 100 U interferon (IFN)-gamma per ml over 3 d and 1 nM phorbol myristate acetate over 24 h resulted in the expression of intercellular adhesion molecule (ICAM)-1 and HLA-DR by infundibular keratinocytes, as determined by immunohistochemistry. Ten nanograms tumor necrosis factor-alpha per ml and 10 ng IL-6 per ml both caused expression of ICAM-1 alone. IL-1alpha had no effect on the expression of ICAM-1 or HLA-DR over 3 d, but addition of 1 ng IL-1alpha per ml over 7 d in culture resulted in hypercornification of the keratinocytes of the infundibulum, apparently brought about by early keratinocyte cornification. These data suggest that the isolated, maintained, infundibulum is a good model for studying the effects of inflammatory cytokines on the infundibulum, and that IL-1alpha acts on infundibular keratinocytes to promote cornification.