Reduced expression of mRNA for transforming growth factor β(TGFβ) and TGFβ receptors I and II and decreased TGFβ binding to the receptors in in vitro-aged fibroblasts

Previous studies have demonstrated that the expression of type I collagen, the most abundant protein in the dermis, is reduced in in vitro-aging fibroblast cultures, but the mechanism controlling the reduction of type I collagen expression is not understood. Recent studies, however, have demonstrated that transforming growth factor β (TGFβ) plays an important role in the regulation of type I collagen expression. The purpose of this study was to investigate the role of TGFβ in downregulation of type I collagen expression in in vitro-aged fibroblasts. We compared the expression of mRNA for α1(I) collagen, TGFβ ,TGFβ type I receptor and TGFβ type II receptor in early- and late-passage fibroblasts by Northern blot hybridizations. The mRNA levels of α1(I) collagen, TGFβ, and TGFβ receptors I and II in late-passage fibroblasts were reduced to 62%, 62%, 59% and 59%, respectively, of those in early-passage fibroblasts. We also compared TGFβ receptor binding in early- and late-passage fibroblasts using receptor binding assays. The affinity of   ¹²⁵ I-TGFβ in late-passage fibroblasts was lower than that in early-passage fibroblasts. These results suggest that the reduction of type I collagen expression in in vitro-aged fibroblasts is regulated by reduced expression of TGFβ and TGFβ receptors I and II and by decreased TGFβ receptor binding ability of the fibroblasts. Received: 13 January 1997     

Ultraviolet B radiation increases steady-state mRNA levels for cytokines and integrins in hairless mouse skin: modulation by topical tretinoin

Chronically sun-damaged human skin has a wrinkled, aged appearance as a result of alterations in the dermal extracellular matrix. Secondary effectors such as cytokines and integrins may mediate the effects of UV radiation on the skin by regulating the synthesis of metalloproteinases and structural proteins including collagen. The aim of this study was to semiquantify the steady-state mRNA levels of interleukin-1α, tumor necrosis factor α, transforming growth factor β, collagenase, stromelysin, collagen, and integrins (α ₁ and α ₂ ) in the skin of hairless mice that were either treated with UV or concurrently treated with UV and topical tretinoin for 5 weeks. Total RNA was extracted from the skin of the mice, reverse transcribed to cDNA, and amplified by the polymerase chain reaction in the presence of ³² P-dCTP using gene-specific primers. Results were normalized relative to glyceraldehyde-3-phosphate dehydrogenase levels. Steady-state mRNA levels of the cytokines and integrins were increased by UV radiation. Concurrent UV and topical tretinoin treatment superinduced the expression of interleukin-1, increased α ₁ and decreased α ₂ integrin expression. Immunofluorescence analysis showed increased dermal localization of β ₁ integrin in UV and tretinoin treated skin. These results suggest that cytokines and integrins may be involved in the mechanism of photodamage. Received: 4 June 1997     

The effects of interleukin-1 and prostaglandin E2 on accumulation of collagen and steady-state levels of proα1(I) collagen messenger RNA in experimental granulation tissue in rats

The effects of human interleukin 1β (IL-1β) and prostaglandin E ₂ (PGE ₂ ) on experimental granulation tissue in rats and on granulation tissue cells in culture were studied. IL-1β and PGE ₂ were injected into subcutaneously implanted sponges during the first 3 days after implantation. The rate of collagen synthesis in fibroblasts was measured as synthesis of protein-bound ³ H-hydroxyproline. The steady-state levels of proα1(I) and proα1(III) collagen chain mRNAs were estimated by Northern transfer analyses. By 7 days postoperatively IL-1β had decreased the hydroxyproline content of granulation tissue. PGE ₂ decreased nonsignificantly the amounts of hydroxyproline, but the steady-state levels of proα1(I) and proα1(III) collagen chain mRNAs were slightly elevated. In IL-1β-treated fibroblast cultures collagen production decreased by 15% and following PGE ₂ treatment by 34% compared with the controls. The latter effect could be abolished by indomethacin. Indomethacin alone stimulated collagen production by 40%. In vivo IL-1 decreases the formation of normal granulation tissue. This effect may be partly due to stimulation of secretion of PGE ₂ . Received: 13 September 1996     

Integrin α2β1 deficiency does not affect contact hypersensitivity

Collagens in the extracellular matrix are thought to play an important role in regulating inflammatory responses by affecting cell adhesion and migration. The contact between collagens and cells is established mainly by α1β1, α2β1 and α11β1integrin receptors. Here, we analyzed the contact hypersensitivity (CHS) reaction in mice that were genetically deficient in the collagen receptor α2β1. Integrin α2β1 is widely expressed and has been suggested to play an important role in mediating inflammatory responses. CHS was induced by applying dinitrofluorobenzene to abdominal skin and challenging with the same reagent on ear skin. Macroscopically and histologically, ear swelling in α2β1-deficient mice did not differ from that in wild-type control mice. Immunohistological detection of infiltrated T lymphocytes, neutrophils and mast cells in inflamed ear skin revealed similar numbers in controls and integrin α2β1-deficient animals. Our results suggest that the adhesive functions of integrin α2β1 are dispensable for the CHS response; they may be compensated for by the collagen receptor α1β1 or other collagen receptors.     

Permeation of hyaluronan tetrasaccharides through hairless mouse skin: an in vitro and in vivo study

Hyaluronan (HA) is a well-known active ingredient for cosmetic and drug applications. However, based on its varying molecular size, HA may have limited skin permeation. Therefore, the aim of the present study was to investigate the in vitro skin permeability of HA tetrasaccharide (HA4). In addition, the effects of HA4 on in vivo skin barrier function were examined. The cumulative amounts of HA4 through stratum corneum (SC)-stripped skin and full-thickness skin after 8 h were 2,109.6 and 0.8 μg/cm², respectively. Furthermore, the cumulative amounts of HA4 permeated after 8 h were 784.4 ng/cm² for a HA4 solution with a pH 4 and 70.0 ng/cm² with a pH 7 on full-thickness skin. Next, the in vivo effects of HA4 on the water content of the SC and transepidermal water loss (TEWL) were investigated. The dorsal skins of hairless mice were irradiated to a UVA dose of 22.3 J/cm²/d, 5 times a week. In the control group, the water content of the SC was decreased and TEWL and epidermal thickness were increased with UVA irradiation than the normal group. However, the water content of the SC was increased in the HA4 group than that of the control group in the non-UVA irradiation groups. In addition, the water content of the SC was increased and TEWL and epidermal thickness were decreased in the HA4 group than those of the control and HA groups. These results suggest that treatment with HA4 improved skin functional recovery after UVA irradiation by skin penetration of HA4.     

Differential response of basal keratinocytes in a human skin equivalent to ultraviolet irradiation

Abstract The human skin equivalent (HSE) provides a convenient model system for studying the cellular responses of basal keratinocytes to UV irradiation. HSEs, constructed by overlaying a collagen-fibroblast matrix with epidermal cells, were raised to an air-liquid interface to promote epidermal differentiation. HSEs were exposed to ultraviolet radiation from a 500-W Hot Quartz Hanovia therapeutic sunlamp, at a total dose of 100 J/m². The HSEs were then frozen every 4 h over a 48-h period and cryosectioned. For each time period, the expression of β₁ integrin and cyclin E, p53, or Bcl-2 were quantified using dual immunolocalization. Basal cells expressing β₁ integrin were divided into two subpopulations, denoted β₁ ^high or β₁ ^low. The proportion of β₁ ^high keratinocytes expressing Bcl-2 and cyclin E increased significantly 4 and 8 h, respectively, after exposure to UV; during the subsequent 16 h, this basal cell subpopulation expressed p53. By contrast, significant numbers of β₁ ^low basal keratinocytes expressed p53, but not Bcl-2. These results suggest that β₁ ^high and β₁ ^low populations of basal epidermal cells in HSEs respond differently to UV irradiation. Received: 30 October 1997     

Autoradiographic mapping of beta-adrenoceptors in human skin

A high density of β ₂ -adrenoceptors has been found in human skin. Using autoradiographic mapping we investigated the distribution of β ₁ - and β ₂ -receptors in normal and diseased human skin. Cryostat sections of human skin obtained at biopsy were incubated with [ ¹²⁵ I]-iodocyanopindolol and nonspecific binding was identified by incubation of adjacent sections with 200 μ M (–)-isoproterenol; β ₂ -receptors were visualized using CGP 20712A and β ₁ -receptors using ICI 118,551 as competing agents. The epidermis was densely labelled with an even distribution throughout all layers. Most of the β-receptors were of the β ₂ -subtype, with practically no β ₁ -receptors. β-Receptors were also localized to eccrine sweat glands, dermal blood vessels, and perivascular inflammatory cells, but there was no labelling of sebaceous glands. Topical glucocorticoids caused an increase in the density of epidermal β-receptors. We conclude that keratinocytes and eccrine sweat glands express high densities of β ₂ -receptors, suggesting that they may have a physiological role in the regulation of these cells. Received: 9 November 1995     

Plasma TM2-PK levels in mycosis fungoides patients

Aerobic glycolysis increases in tumor cells and pyruvate kinase (PK) is one of the key enzymes involved; PK exists in different isoforms in various tissues. Tumor M2-PK (TM2-PK) is one of these isoforms and its expression has been observed in various tumor cells, including lymphocytes, and in lymphoproliferative disorders. The present study aimed to compare plasma levels of TM2-PK and serum levels of two established markers of various lymphoproliferative disorders—lactate dehydrogenase (LDH) and β-2 microglobulin, and to evaluate the role of TM2-PK in drug monitorization and disease activity in mycosis fungoides (MF) patients. The study included 27 MF patients and 46 healthy controls. Among the MF patients, 18 were stage IA, 6 were stage IB, 1 was stage IIA, and 2 were stage III. Plasma TM2-PK, and serum LDH and β-2 microglobulin levels in the patients and controls were measured using the ELISA technique, a kinetic method, and a chemiluminescent assay, respectively. Measurements were repeated in the patient group posttreatment. Median levels of TM2-PK, LDH, and β-2 microglobulin level in the MF patients were 22 U mL⁻¹, 375 U L⁻¹, and 1,831 ng mL⁻¹, respectively. TM2-PK and β-2 microglobulin levels did not significantly differ between the MF patients and controls; however, LDH levels were significantly higher in the MF patients. TM2-PK levels in 17 of the MF patients that were in remission did not significantly differ from their pre-therapy levels. Based on a cut-off point of 17.5 U mL⁻¹, the sensitivity and specificity of TM2-PK for diagnosing MF were 55.6 and 60.9%, respectively. β-2 microglobulin was the most sensitive marker for diagnosing MF (63%), while LDH was the most specific marker. Furthermore, the sensitivity of TM2-PK increased when it was analyzed in combination as parallel tests with LDH and β-2 microglobulin (86%), while the specificity was measured as 32%. In serial analysis, the specificity was increased to 98%, while the sensitivity was 5%. Statistically significant agreement in diagnosing MF was also noted between TM2-PK and LDH levels. TM2-PK may not be a useful marker for MF, especially in early-stage patients, because it proliferates slowly. We think that TM2-PK levels should be investigated in advanced-stage MF or in other types of cutaneous T-cell lymphomas; in particular, in combination with other established markers.     

Ginsenoside Rg1 protects human fibroblasts against psoralen- and UVA-induced premature senescence through a telomeric mechanism

This study was aimed to investigate the protective effects of ginsenoside Rg1 on 8-methoxypsoralen(8-MOP)/Ultraviolet A (UVA)-induced premature senescence in human fibroblasts, and the underlying mechanism. We established a stress-induced premature senescence model by 8-MOP/UVA irradiation. The aging condition was determined by histochemical staining of senescence-associated β-galactosidase (SA-β-gal). Relative telomere length was calculated by the ratio of the amount of telomere DNA versus single copy DNA by real-time polymerase chain reaction, and protein levels of p-P53, p21^WAF−1 and p16^INK−4a were estimated by Western blotting. Compared with the 8-MOP/UVA treatment group, we found that the irradiated fibroblasts pretreated with ginsenoside Rg1 demonstrated a decrease in the expression of SA-β-gal, a downregulation in the level of senescence-associated proteins, and a deceleration in telomere shortening. Taken together, these results suggest that ginsenoside Rg1 significantly antagonizes premature senescence induced by 8-MOP/UVA in fibroblasts.     

2-kDa hyaluronan ameliorates human facial wrinkles through increased dermal collagen density related to promotion of collagen remodeling

Background/Aims

Hyaluronan (HA) oligosaccharides are involved in several biological processes, primarily collagen remodeling and wound healing. Collagen remodeling is retarded in aging skin and causes wrinkles. The aim of this study was to evaluate the effect of 2-kDa HA oligosaccharides (HA2k) on wrinkles by permeation through the stratum corneum and promotion of collagen remodeling.

Methods

A 3D skin model and excised human skin were used to evaluate the permeation of fluorescein-labeled HA2k. The effect of HA2k on collagen metabolism was evaluated by measuring the protein level of type 1 pro-collagen (COL1A1) and matrix metalloproteinase-1 (MMP-1) in the 3D skin model. 0.1% HA2k solution and vehicle control was applied to the human forearm for 8 weeks to evaluate dermal collagen density. To evaluate the effect of HA2k on depth of facial wrinkles, a randomized controlled trial was conducted with 0.1% HA2k lotion and vehicle lotion for 8 weeks.

Results

HA2k was confirmed to permeate through the stratum corneum by fluorescent microscopy. Both COL1A1 and MMP-1 were upregulated by HA2k application in a 3D skin model culture. The collagen density was higher for the HA2k-treated forearm than for the vehicle control-treated forearm after 4 weeks. The maximum wrinkle depths in the nasolabial fold and crow's feet area were significantly shallower in the HA2k lotion group than in the control group.

Conclusion

HA2k permeated the stratum corneum, activated collagen synthesis and degradation simultaneously, and ameliorated wrinkles.     

Antioxidant defence mechanism of the skin against UV irradiation: Study of the role of catalase using acatalasaemia fibroblasts

To clarify the role of catalase, an antioxidant enzyme, in response to UV irradiation, we compared the effects of irradiation on cytotoxicity, activities of antioxidant enzymes, total glutathione concentrations, lipid peroxidation and the rate of collagen synthesis in skin fibroblasts from a patient with acatalasaemia and in those from a normal individual. The cells were irradiated with UVA (6 and 12 J/cm² or UVB (0.5 and 1 J/cm²). Cell survival curves after UV irradiation were similar in cells from both subjects. Although superoxide dismutase activity in acatalasaemia cells was higher than in the control cells before irradiation, after irradiation the activity decreased in acatalasaemia cells (76% with 12 J/cm² UVA, 47% with 1 J/cm² UVB), but remained unchanged in control cells. Total glutathione concentrations also decreased in acatalasaemia cells (60% with 12 J/cm²) in response to UVA irradiation, but remained unchanged in control cells. Lipid peroxidation did not increase significantly in either cell type. The rate of collagen synthesis decreased to a similar extent in response to UV exposure in the two cell types (60–80% with 8.2 J/cm² UVA, 40–50% with 10 mJ/cm² UVB). We conclude from the results of cytotoxicity and lipid peroxidation that although acatalasaemia cells were killed by hydrogen peroxide at low concentrations with a single UV exposure, catalase functions only to a small degree as an antioxidant enzyme. There remains the possibility, however, that a deficiency of catalase may chronically damage the skin resulting in a reduced defence function of Superoxide dismutase and glutathione with repeated exposures to UV, which is becoming more common in our daily life.     

Simultaneous analysis of [3H]-thymidine uptake and α1(I) procollagen mRNA expression in systemic sclerosis skin fibroblasts – an in situ hybridization study

Abstract Heterogeneity of DNA synthesis and collagen synthesis has been reported in skin fibroblasts from systemic sclerosis (SSc) patients. The uptake of [³H]-thymidine and expression of α1(I) procollagen mRNA by cultured skin fibroblasts from four normal controls and four SSc patients was analyzed simultaneously. The grains overlying the cytoplasm representing α1(I) procollagen mRNA and overlying the nucleus representing [³H]-thymidine uptake were counted using computer-aided image analysis. The results were analyzed statistically. Procollagen mRNA expression by SSc fibroblasts was significantly greater than by control fibroblasts (P < 0.01). The distribution curve of [³H]-thymidine uptake showed two peaks representing low- and high-uptake cells. Significantly more SSc fibroblasts than control fibroblasts showed high [³H]-thymidine uptake (P < 0.05). The number of SSc fibroblasts expressing low amounts of α1(I) procollagen mRNA was significantly lower than the number of control fibroblasts (P < 0.05). [³H]-thymidine uptake by SSc fibroblasts expressing high amounts of α1(I) procollagen mRNA was significantly lower than by those expressing low amounts (P < 0.05). These results indicate that elevated DNA synthesis and elevated collagen mRNA synthesis in SSc skin fibroblasts are due to different clones with high DNA-synthesizing ability and high collagen-producing ability. Received: 5 October 1999 / Revised: 20 December 1999 / Accepted: 23 December 1999     

Inhibition of collagen expression by azelastine hydrochloride in cultured skin fibroblasts from normal individuals and scleroderma patients

The effects of azelastine hydrochloride on cell proliferation and collagen synthesis in cultured human skin fibroblasts were studied. Azelastine inhibited cell proliferation during proliferating cell phases. Azelastine was found to inhibit collagen synthesis without altering cell proliferation during quiescent phases. It did not alter the ratio of type I to III collagen synthesis. Northern blot analysis of collagen chain mRNAs revealed that the levels of α₁ (I), α₁ (III) and α₁ (VI) mRNAs were reduced by azelastine treatment, whereas the level of α₂ (VI), α₃ (VI) mRNAs were not significantly changed. These results suggest that azelastine modulates collagen synthesis at a pretranslational level. Azelastine inhibited collagen synthesis in fibroblasts from scleroderma patients to the same extent as in normal skin fibroblasts. This drug may be useful in the treatment of fibrotic diseases.     

Synergy of GHK-Cu and hyaluronic acid on collagen IV upregulation via fibroblast and ex-vivo skin tests

Introduction

GHK-Cu and HA are two commonly used skin care ingredients, both of which were reported to enhance collagen synthesis. This work aims to investigate their co-effect on collagen regulation.

Materials and Methods

In cell experiments, human dermal fibroblasts were treated by a series of GHK-Cu and HA combinations, and the expressions of collagen I, IV, and VII were measured by qRT-PCR. The best formula screened out from cell experiments were further studied by ex-vivo skin model, and the content of collagen IV was quantified by immunofluorescence method.

Results

The combination GHK-Cu and HA was found to promote the generation of collagen I, IV, and VII. Especially, they form a synergy on collagen IV. At the ratio of 1:9, GHK-Cu and LMW HA deliver the strongest effect to elevate collagen IV synthesis by 25.4 times in cell test and 2.03 times in ex-vivo skin test.

Conclusion

The co-effect of GHK-Cu and HA was revealed. Their synergy brings an insight to anti-aging technology: choosing proper molecular weight of HA and managing its ratio with GHK-Cu could enhance DEJ health via stimulating collagen IV synthesis.     

Effect of repeated ultraviolet irradiation on skin of hairless mice

Summary The effect of repeated UV-irradiation on mechanical and biochemical parameters was studied in skin of hairless mice. UV-A irradiation for a period of 1 h daily over 8 weeks caused only a slight increase in skin thickness and a decrease in ultimate strain. The changes induced by UV-B and C, however, were quite remarkable.Skin thickness was increased depending on the daily dose exposure time (15–90 s at an irradiation rate of 20 mW/cm² UV-B and A and of 14 mW/cm² UV-C) and the duration of treatment (1–6 weeks). Ultimated load, tensile strength and modulus of elasticity showed an increase following medium dosages after 1 and 2 weeks, however, a decrease after high dosages and longterm treatment. Ultimate strain was found to be the most sensitive parameter being decreased depending on exposure time and duration of treatment. Insoluble collagen and total collagen were decreased after long-term treatment thus being correlated with the mechanical parameters. The elastin content was only barely influenced and not correlated with the mechanical data, e.g. the modulus of elasticity. Thus, a favourable effect of short-term treatment with low doses of UV-irradiation of mechanical parameters of skin could be demonstrated. Long-term treatment with relatively high doses of UV-B, however, resulted in unfavourable effects, whereby first ultimate strain, then ultimate load, modulus of elasticity and tensile strength were decreased.     

Percutaneous penetration of dipyrithione in man: Effect of skin color (race)

A single chemical dose of dipyrithione (2,2′-dithiobispyridine-l, 1′-dioxide), 4 or 12 μ/cm² (containing 1 μCi of 2–6-¹⁴C material), was applied to the ventral forearm (intact skin, methyl alcohol vehicle [MeOH]), forehead (intact and stripped skin; cosmetic cream and MeOH vehicle), and scalp (intact and stripped skin; shampoo and MeOH vehicle) of human (white and black) volunteers. The urinary excretion of ¹⁴C was measured over the 7-day study period. Percutaneous penetration data were corrected for incomplete urinary recovery using data from intravenous studies (Wedig et al¹). The results obtained in this study suggested: (1) means from the complete analysis indicated 34% less (p < 0.02) absorbed by blacks; (2) less was absorbed by blacks than whites when seven out of seven means were compared within groups (p < 0.02); (3) a difference (p < 0.03) in penetration, blacks 47% lower than whites, between a cosmetic cream vehicle vs MeOH on the forehead; (4) on the scalp (MeOH vs shampoo vehicle) more was absorbed using MeOH (p < 0.02); (5) no evidence of a significant difference in penetration was noted when (a) the various anatomic sites were compared or (b) intact skin was compared with stripped skin.     

Topical bioavailability of triamcinolone acetonide: effect of dose and application frequency

The application frequency of topical corticosteroids is a recurrently debated topic. Multiple-daily applications are common, although a superior efficacy compared to once-daily application is not unequivocally proven. Only few pharmacokinetic studies investigating application frequency exist. The aim of the study was to investigate the effect of dose (Experiment 1) and application frequency (Experiment 2) on the penetration of triamcinolone acetonide (TACA) into human stratum corneum (SC) in vivo. The experiments were conducted on the forearms of 15 healthy volunteers. In Experiment 1, single TACA doses (300 μg/cm² and 100 μg/cm²) dissolved in acetone were applied on three sites per arm. In experiment 2, single (1 × 300 μg/cm²) and multiple (3 × 100 μg/cm²) TACA doses were similarly applied. SC samples were harvested by tape stripping after 0.5, 4 and 24 h (Experiment 1) and after 4, 8 and 24 h (Experiment 2). Corneocytes and TACA were quantified by UV/VIS spectroscopy and HPLC, respectively. TACA amounts penetrated into SC were statistically evaluated by a paired-sample t-test. In Experiment 1, TACA amounts within SC after application of 1 × 300 μg/cm² compared to 1 × 100 μg/cm² were only significantly different directly after application and similar at 4 and 24 h. In Experiment 2, multiple applications of 3×100 μg/cm² yielded higher TACA amounts compared to a single application of 1 × 300 μg/cm² at 4 and 8 h. At 24 h, no difference was observed. In conclusion, using this simple vehicle, considerable TACA amounts were retained within SC independently of dose and application frequency. A low TACA dose applied once should be preferred to a high dose, which may promote higher systemic exposure.     

Reconstruction of basement membrane in skin equivalent; role of laminin-1

Abstract To reconstruct the basement membrane in a skin equivalent, the epidermodermal interface was coated with porcine type IV collagen and mouse laminin-1 at various ratios before keratinocyte seeding. Laminin-1, a component of the basement membrane, induced massive infiltration of keratinocytes into the dermal equivalent, while type IV collagen induced discrete demarcation between dermal and epidermal compartments without any infiltrating cells. Immunohistochemical staining indicated that the laminin-induced infiltrating cells expressed endogenous type IV collagens at the cell periphery, which were not incorporated into the basement membrane structure. The infiltrating cells did not express fibronectin receptor α₅β₁ integrin but showed MMP-9 secretion and cell surface associated MMP-2. However, when laminin-1 was preincubated with type IV collagen, laminin-1-induced keratinocyte infiltration as well as MMP-9 induction were almost completely suppressed to basal levels. Therefore, replenishment of the type IV collagen lattice seemed to cause laminin-stimulated cells to anchor to the lattice, in a similar manner to the basal cells on the basement membrane of normal skin. Our study suggests that the molar ratio of basement membrane components may determine the behavior of basal cells within the wound healing microenvironment, which is probably regulated either by extracellular matrix deposition or degradation. Received: 24 October 2000 / Revised: 9 February 2001 / Accepted: 31 March 2001     

IL-1 and IL-1 receptor antagonist regulation during keratinocyte cell cycle and differentiation in normal and psoriatic epidermis

Abstract Changes in the levels of IL-1 (IL-1α, IL-1β, and its receptor antagonist, IL-1RA) occur upon keratinocyte differentiation in vitro and are associated in vivo with abnormal differentiated and hyperproliferative states of psoriatic keratinocytes. A flow cytometric procedure, capable of detecting changes in the intracellular levels of IL-1, was used to determine whether intracellular IL-1/IL-1RA levels in psoriatic and normal keratinocytes alter during in vivo differentiation and the cell cycle. Increases in the IL-1RA levels and IL-1α levels were observed as both normal and psoriatic keratinocytes differentiated from basal stem cells (β₁ integrin⁺, small size) into transient amplifying cells (TAC; β₁ integrin⁺, large size). Upon further differentiation (β₁ integrin^–, large size) both IL-1RA and IL-1α levels dropped. However, while psoriatic IL-1β levels increased as cells differentiated into TACs, little change occurred in the IL-1β levels of normal keratinocytes during differentiation. Changes in IL-1/IL-1RA levels were also detected as keratinocytes progressed through the cell cycle. Within the basal stem cell population of both normal and psoriatic keratinocytes, the IL-1α and IL-1RA levels increased between G0/G1 and S but not between S and G2/M. However, psoriatic basal keratinocyte IL-1β levels differed from those of normal keratinocytes by showing no increase between S and G2/M. The IL-1/IL-1RA levels of normal TAC increased throughout the cell cycle. However, in psoriatic TAC, a slight decrease in IL-1α and IL-1RA levels was observed between G0/G1 and S followed by a delayed increase between S and G2/M. IL-1β levels in psoriatic TAC varied little throughout the cell cycle. Thus, we were able to detect precisely the regulation of IL-1/IL-1RA intracellular levels during the keratinocyte cell cycle and differentiation, showing notably decreased IL-1β upregulation in psoriatic keratinocytes progressing through the cell cycle. Received: 15 July 1996