effects of glucocorticoids on cultured skin
DESCRIPTION
Effects of Glucocorticoids on Cultured SkinTRANSCRIPT
Review
Effects of Glucocorticoids on Cultured Skin
Anti-inflammatory steroids, which are probably the most frequently used drugs in dermatology, have many beneficial properties, but long-term use of potent ste- roids can lead to undesirable side effects, for example skin atrophy. In skin atrophy induced by glucocor- ticoids, both the epidermis and the dermis are involved. The thinning of these layers might be due to the antimito- tic andlor antisynthetic action of steroid. It is not clear from the literature which of these skin layers is more strongly involved in the development of skin atrophy. Since both layers are normally always present, steroid- induced metabolic changes in one of them might influ- ence the metabolic processes in the other.
All of the approaches used so far to study skin atrophy, eg, histological investigation, measurement of changes in skin thickness, studies in animals, have some advan- tages and disadvantages. The use of cultured keratino- cytes and fibroblasts originating from human skin (Fig. 1) i s an in vitro model that offers a system in which the effects of glucocorticoids on various types of cell can be studied separately. The main advantages of this system are that the studies can be performed under well- controlled conditions, and cells originating from various donors can be used.
Human skin fibroblasts in culture have become an important aid in the investigation of genetic defects and for understanding of the biochemical nature of the growth, aging, and neoplastic transformation of cells. Let me mention some examples of studies done with fibroblasts.
The fibroblasts originating from patients with heritable disorders of connective tissue, eg, patients with the Ehlers-Danlos (E-D) syndrome or osteogenesis imper- fecta (01) continue to show the abnormal behavior, ie, altered collagen metabolism) in cell culture. It has been shown by various investigators that the abnormalities
Fibroblasts
Addres, for reprints: Maria Ponec, Ph.D., University Hospital, Department of Dermatology, Rijnsburgerweg 10, 2333 AA Leiden, The Netherlands.
and Keratinocytes
MARIA PONEC, PH.D.
From the Department of Dermatology, University Hospital, Leiden,
The Netherlands
can occur at various levels of collagen metabolism, for instance, defects of biosynthesis (E-D type IV, 011, post- translational modification (E-D, type VI, OI), secretion of procollagen (E-D, type IV C), and extracellular alterna- tions of collagen (E-D types VII and V) (for a review, see Bornstein and Byers').
Another example of the usefulness of this in vitro model i s provided by studies on the aging process. Hay- flick2 showed in 1965 that cultured fibroblasts have a finite capacity for replication and that this capacity is age-related. This finding has since been confirmed by many investigators. Rheinwald and Green3 showed that human cultured keratinocytes, too, have a restricted lifetime of replication. These authors also showed that there is an age-related decrease in the number of genera- tions and in plating efficiency. Furthermore, age-related alterations in the cell-membrane and extracellular- matrix composition have been found in cell cultures as well as in vivo. The response to glucocorticoids changes also during aging, partially due to a decrease in the number of specific glucocorticoid binding sites6 The decreased sensitivity to glucocorticoids has been also observed in aged animals.'
Glucocorticoids have been reported to prolong the life span of cells in culture.6 Furthermore, the effects were more pronounced when the glucocorticoid was adminis- tered to cells during early passages, and decreased pro- portionally with the age of the culture at the time when the steroid was administered.'
The above-mentioned examples illustrate the value of in vitro experiments; however, great caution must al- ways be exercised with respect to the extrapolation of the data obtained in in vitro studies to the in vivo situa- tion, where numerous humoral factors originating from
11
12 INTERNATIONAL IOURNAL OF DERMATOLOGY January-February 1984 Vol. 23
FIL. 1 . ( A . lerf) Confluent culture of human foreskin fibroblasts. (For experimental data 5ee reference 24.) (5. rlght) Confluent culture of human foreskin keratinocytes coculfured with 3T3 feeder layer. (For experimental data see references 3 and 12 )
other tissues are possibly present. It is hard to simulate exactly this situation in vitro, and this can influence the metabolic processes of the particular cell type under study.
Many of the papers in the literature deal with the physiological and biochemical effects produced by glucocorticoids. It is currently thought’ that most of the inhibitory and stimulatory actions of the glucocorticoid hormones are initiated by the binding of the steroid to specific glucocorticoid receptors, which are located in the cytoplasm, and by formation of the receptor-steroid complex. After a process called “activation,“ the steroid receptor complex i s translocated to the nucleus and binds to the chromatin, causing a modulation of genomic expression probably at the level of the forma- tion of specific mRNAs. The translation products of these mRNAs then mediate the glucocorticoid response.
From many aspects of the interactions of glucocorti- coids with living cells the following will be discussed: the penetration of glucocorticoids into cells and the binding to the receptor, as well as effects of glucocor- ticoids on cell proliferation, the synthesis of matrix com- ponents, and prostaglandins.
Penetration of Steroids
The mechanism by which steroids enter the cell is not clear yet. For many types of cell, a very rapid uptake’has been reported. It is assumed that the steroid entry into most cells occurs by passive diffusion, but for some cells, a carrier-mediated process has been reported as well (for a review, see Raos).
A current study in our laboratory has shown (unpub- lished data) that the entry of various glucocorticoids into cultured human skin fibroblasts is indeed a very rapid process. The steady state was reached within 1-2 min-
utes. The uptake of glucocorticoids by cultured keratinocytes proceeded somewhat more slowly, and a steady state was not observed even after one hour of incubation. In spite of the fact that for both cell systems the ratio of the steroid concentration (ci/co) within the cell to that outside the cell is greater than one for all glucocorticoids studied, we assume that glucocorticoids enter these cells by simple, and not by facilitated, diffu- sion. We make this assumption because the uptake process was not saturable, the amount of steroid taken up by the cell increased linearly with increasing steroid concentration in the medium (from 1 O-’ to 1 0-6M). No competition was found between various steroids for the cellular uptake, even when an excess of one steroid was present. The preincubation of cells with a high concen- tration ( ~ O - ~ M ) of the same or another steroid did not affect the rate of steroid uptake at a concentration of
M. Lowering the temperature to 0 C slowed down the rate of uptake, but the ci/co ratio still remained much higher than one. Similar findings were recently reported by Giorgi and Steing for hamster fibroblasts and rat hepatoma cells.
Increasing of the osmolarity in the medium causes a reduction of the cellular water space. The amount of steroid taken up by the cell was only slightly affected by decreased cellular water space, which suggests that the steroid accumulates in some “nonwater space,” eg, the cell membrane. The amount of steroid that accumulates in the cell and also in this nonwater space of the cell has been found to be a function of the lipophilicity of the steroids. For most steroids, it holds that the greater the lipophilicity the greater the uptake (Table 1 ) .
On the basis of these results, i t is conceivable that the steroid simply first dissolves in the lipid material of the membrane and then becomes bound to the intracellular receptor. It i s of interest to mention that when a steroid
No. 1 EFFECTS OF GLUCOCORTICOIDS Ponec 13
was applied in vitro to epidermis (including the outmost horny layer, which forms the main barrier of penetration) in an ethanolic solution, the reverse relationship was found for the penetration of steroid through the horny layerlo: the greater lipophilicity, the lower the penetra- tion rate. The penetration rate through the horny layer was much slower than that through the cell membrane of keratinocytes and of fibroblasts. These findings con- cerning penetration through the horny layer on the one hand and into epidermal cells and fibroblasts on the other illustrate the complexity of steroid action in vivo. In vivo, the steroid must penetrate the stratum corneum and upper layers of epidermis before it reaches its "site of action." Once there, it must enter the cell and bind to the intracellular receptor. This last step will be discussed in the following section.
Clucocorticoid Receptor
lntracellular receptor molecules that bind steroids occur in a number of tissues. Cultured human skin fibro- blastsll and keratinocytes** have in their cytoplasm mac- romolecules that bind glucocorticoids with high affinity and specificity. The availability of tritiated glucocor- ticoids of high specific activity has made it possible to detect the binding of steroids to these macromolecules. Studies done in our laboratory have shown that, at 0 C and with the use of 3H-triamcinolone acetonide, H- dexamethasone, or H-hydrocortisone as ligand, specific binding sites in the cytosol of cultured human foreskin keratinocytes and fibroblasts can be dernon- strated. These sites became saturated at steroid concen- trations between 20 and 50 nM, ie, the same range as found in other cells and tissues. The affinity of the above-mentioned steroids for the receptor (expressed as K,,,,,) was in the range 3-25 nM, and the number of receptor sites ranged from 100-300 fmoles/mg cytosolic protein. The binding constants were similar to those found by Epstein and B ~ n i f a c ' ~ - ~ ~ and Leiferman et all5 in viable human epidermis and dermis as well as in other cell systems. Epstein and BonifacI4 also demonstrated the binding of glucocorticoid receptor complex of human epidermis to the nucleus. Leiferman et al l5 found great differences in the number of receptor sites per cell in whole skin originating from various anatomical sites, eg, for the abdomen 294, for the face 3,200, and for the foreskin 5,133 sites per cell. This difference might be related to the clinically observed difference in the sensi- tivity of various areas of the body to glucocorticoids. Leiferman et al l5 also found that the number of receptor sites per cells was almost 10 times higher in epidermis than in dermis (2057 and 243 sites/cell, respectively). In cultured cells, when the number of receptor sites is ex- pressed per mg cytosolic protein, the difference between
TABLE 1. Uptake of Clucocorticoids by Cultured Human Skin fibroblasts and Keratinocytes
C,IC.' Steroid Fibroblasts Keratinocytes Log Pt
Hydrocortisone 2.1 1.9 1.52 Dexamethasone 9.7 10.0 1.81 Triarncinolone acetonide 7.8 12.0 2.38 Clobetasol propionate 33.7 39.7 2.99 Clobetasol butyrate 29.0 34.6 3.75
The ratio of the steroid concentration in the cell (C,) to the extracel- lular concentration (C,,]. The confluent cultures were incubated at 37 C in the presence of increasing concentrations of radiolabeled steroids for 5 min (fibroblast cultures) and for 60 min (keratinocyte cultures), after which the cells were washed 5 times with ice-cold phosphate buffer, and the radioactivity in the cells and in the medium was determined. The cell water volume was estimated by measuring the equilibrium uptake of 3-O-methyl-D-gl~cose.~
t Log P was determined as log of the partition cwfficient between n a t a n o l water by high performance liquid chromatography (HPLC).
the number of receptor sites in keratinocytes and in fibroblasts was small.
How does the steroid bind to the receptor? Wolff et al l6 performed thermodynamic studies to investigate this problem, and they postulated that the interactiori be- tween the steroid and receptor is mainly hydrophobic; that is, the displacement of water molecules from strongly hydrated steroid and receptor i s a principal driv- ing force of the binding. These authors suggest a model in which the steroid i s enveloped by the receptor. Vari- ous substituents on the steroid molecule can signifi- cantly influence the binding affinity of the steroid for the receptor, ie, by changing the lipophilicity, electron den- sity, and conformation of the molecule. Wolff et all6 reported that the following structural features of the ste-
TABLE 2. Effect of Esterification of Hydrocortisone al C,, and C,, on the Relative Binding Affinity
to C/ucocortkord Receptor in Cytosol of Cultured Human Keratinocytes
Steroid IC,LnM)' ~~
Hydrocortisone Hydrocortisone-1 7-acetate Hydrocortisone-1 7-propionate Hydrocortisone-1 7-butyrate Hydrocortisone-1 7-valerate Hydrocortisone-2 1 -acetate Hydrocortisone-2 1 -propionate Hydrocortisone-2 1 -butyrate Hydrocortisone-2 1 -valerate
125 145 75 45 37
1630 670 355 460
Values are mean of three experiments (see also Table 3)
14 INTERNATIONAL JOURNAL OF DERMATOLOGY January-February 1984 Vol. 23
21;H20" c = o
18 2 0 -
0 FIG. 2 The structure of hydrocortisone
roid account for the resulting receptor affinity: surface area, A-ring conformation, size of the 9a substituent, and the polarity of the steroid. The binding of steroid to the receptor can also be influenced by the substituents on C,, and C,,, which can significantly alter the rota- tional freedom of the 17p side chain.
Our recent studies on the relationship between struc- ture and binding, done with a cytosol fraction of cul- tured human keratinocytes, revealed the importance of a number of substituents on the steroid molecule.
According to the literature, binding of steroids to the glucocorticoid receptor only takes place i f the steroid molecule distinguishes itself by at least the following substituents: a double bond at C, and C5, and 3.20-keto and 11/3-hydroxyl groups as in hydrocortisone16 (Fig. The introduction of the C,-C, double bond, the presence of the 9a F atom and of the 1 1 p hydroxyl group, and the esterification of the 17a hydroxy group caused an in-
TABLE 3. Relative Binding Affinity of Various Topically Applied Clucocortiroids to the Clucocorticoid Receptor in
Cytosol of Cultured Human Keratinocytes
Steroid IC,(nMY
Hydrocortisone Hydrocortisone-1 7-butyrate Triamcinolone acetonide Clobetasone-17-butyrate Clobetasal- 1 7-propionate Betamethasone-1 7-valerate
130 36 28 24 16 10
I&, is the mean concentration at which the 50% displacement of specifically bound 3H-dexamethasone took place. The aliquots of the cytosol fraction of cultured keratinocytes were incubated with radiolabeled 3Hdexamethasone (at 3.5 x MI and in the absence or in the presence of increasing concentrations of nonlabeled steroid (10-n-10-6 MI. The displacement of specifically bound dexamethasone by nonlabeled steroids was measured as described in detail elsewhere.'l
crease of the affinity of the steroid for the receptor, whereas esterification of 21 -hydroxy group led to a de- crease of this affinity. The effect of esterification at the Cii and C,, positions was dependent on the length of the ester. Affinity increased with increasing length. For all 21 esters, however, the affinity was found to be less than that of 21 alcohol (Table 2).
As could be expected, cortisone-a steroid with a keto group in the C,, position-showed no affinity for the receptor. Despite the presence of the keto group on C,, , the synthetic glucocorticoid clobetasone-17- butyrate displayed a marked affinity for the receptor, comparable with that of triamcinolone acetone (Table 3). The negative effect of the C,, keto group was found to be counteracted by other substituents in the molecule, such as a fluor atom in the 9a. butyrate in the 17a. and CI in the 21 position. These findings indicate the impor- tance of studies on the structure-binding relationship for the development of new topical steroids.
The glucocorticoids used in clinical practice vary as to therapeutic potency, which for most of the steroids we have studied corresponds with their binding affinities to the cytosolic receptor (Table 3).'* Hydrocortisone, the clinically least potent steroid, showed the lowest affinity for the receptor. Other steroids such as hydrocortisone- 1 7-butyrate, dexamethasone, and triamcinolone acetonide, showed much higher affinity, followed in the order by clobetasone butyrate, clobetasol-17- propionate, and betarnethosone-17-valerate.
The whole process of the steroid-receptor interaction has recently been discussed in detail in a review by johnson et al." As pointed out, most of the glucocor- ticoid actions are initiated by the binding of the steroid to glucocorticoid receptors. Following a process called activation, the receptor-steroid complex binds to the nucleus. The binding of steroid to the receptor is proba- bly accompanied by phosphorylation of the receptor, the activation of the receptor-steroid complex by its de- phosphorylation. The activated receptor-steroid com- plex is then translocated to the nucleus and binds to the chromatin, which leads to changes in chromatin con- formation. This i s probably accompanied by phosphory- lation of certain nuclear nonhistone proteins, which would alter the number of initiation sites for RNA polymerase, followed by changes in the transcription of specific mRNAs. Compared with the number of cyto- plasmic receptor sites, the number of acceptor sites in the nucleus has been found to be in excess. The direction and the magnitude of the change in the number of RNA polymerase initiation sites are also regulated by the activity of other hormones, which have also been shown to affect the phosphorylation of the nuclear nonhistone proteins, not always the same set as those affected by glucocor- ticoids. The final effect of glucocorticoids is, therefore,
EFFECTS OF CLUCOCORTICOIDS Ponec 15 No. 1
among others dependent on the presence or absence of other hormones in the studied system.
Besides the action of the steroid-receptor complex via a nuclear pathway, it is possible that direct receptor- mediated actions of steroid on other cell substrates take place. Some effects occur so rapidly that involvement of the already mentioned transcriptional mechanisms is highly unlikely. Johnson et all7 thought that the rapid effects of glucocorticoids on prostaglandin production they observed were probably attributable to inhibition of a Ca2+ influx or of the c-AMP-dependent protein kinase, an effect that could produce major changes in the cellular metabolism. Also, rapid effects of glucocorticoids on the uptake of glucose and amino acid have been observed by various authors. All these effects were observed within a few minutes after glucocorticoid administration, and probably occur via the non-nuclear pathway, since re- sponse via the nuclear pathway would proceed much more slowly.
Effects of Clucocorticoids on Cell Proliferation
Glucocorticoids have been repeatedly reported to act as modulators of cell proliferation. In vitro, a wide range of cell types display some reaction to the steroid hor- mones, from stimulation of cell growth to severe growth retardation and, in some cases, even cell death and lysis (for a review, see Cristofalo and Rosner6).
Glucocorticoids can either inhibit or stimulate DNA synthesis and cell growth in fibroblasts, both in primary culture and in established cell lines, depending upon the origin of the cells and the culture conditions applied. Effects of glucocorticoids on the proliferation of human skin fibroblasts are summarized in Table 4, from which it can be seen that both inhibition and stimulation of cell
I proliferation by glucocorticoids have been reported. Many factors could be responsible for discrepancies be- tween results obtained in different laboratories. Some examples may be mentioned.
The origin of the fibroblasts may influence the effect of glucocorticoids on cell proliferation, because cultures of fibroblasts from whole-skin samples were recently shown to contain a heterogenous cell population.:'O."' The cells originating from papillary dermis grew faster and longer than cells from reticular dermis, and one would therefore expect differences in the sensitivity of these cells to glucocorticoids.30 Many of the discrepan- cies between the results of various investigators with re- spect to effects of glucocorticoids on cell proliferation might arise from differences in the cell populations used in different laboratories, eg, cells from fetal and adult skin and from different parts of the body. Furthermore, our studiesR2 have shown that steroid-induced growth inhibition is dependent on cell density. Marked growth inhibition occurred in cultures with a low cell density,
and the degree of inhibition decreased with increasing cell density. Dense cultures displayed no effect of glucocorticoid or even a slight stimulation of cell prolif- eration. JohnsonlR investigated the degree to which the biological response to glucocorticoids is sensitive to the metabolic state of the cell. When hydrocortisone was added to human skin fibroblasts without simultaneous renewal of the medium (nutritient-depleted medium), H-thymidine incorporation into DNA was inhibited,
whereas stimulation was observed when medium re- newal and steroid addition were done simultaneously ("enriched medium"), and these effects were also re- flected in a decrease or increase, respectively, in the number of RNA polymerase initation sites. These authors also showed that glucocorticoid-induced phosphoryla- tion of nuclear nonhistone proteins might be influenced by other hormones, the level of which might depend on whether "fresh" or "depleted" medium is present, and thus affect the final action of glucocorticoids. Further- more, the direct effect of glucocorticoids on the Caz+ flux, which influences the metabolic state of the cell, might differ under different culture conditions. Saarni and Tammi33 studied the role played by the duration of exposure to glucocorticoids. Only relatively long expo- sure (84 hr) to a low concentration of hydrocortisone
M inhibition occurred after only 24 hr.
The type (newborn or fetal calf serum, human serum) and amount of the serum added to the culture medium can also modify the effect of a glucocorticoid on cell p r ~ l i f e r a t i o n , ~ ~ because serum contains a variety of mitogenic factors, eg, epidermal growth factor (EGF), and fibroblast growth factor (FGF), whose level can dramatically alter this effect. In human skin fibroblasts, cortisol was found to stimulate the incorporation of RH- thymidine into DNA when EGF is present. EGF had no effect on the number of receptor sites or the affinity of the steroid for the receptor.
Glucocorticoids were found to enhance the binding of EGF to surface receptors on cultured human skin fibro- blasts.6-35 This might be even one of the mechanisms by which the steroids increase the proliferation rate. In other cell systems, various effects of growth factors and glucocorticoids have been reported. For example, EGF was found to stimulate cell division in confluent quies- cent 3T3 cells. This effect was counteracted by glucocor- ticoids; however, FGF, in combination with glucocor- ticoids, gave a further enhancement of cell division,Rfi which suggests that different growth factors may trigger different intracellular events. Cospodarowicz et a13i showed that fibroblasts could only proliferate in serum- free medium when dexamethasone and FGF were sirnul- taneously present. The growth of epithelial cells was also enhanced in the presence of EGF and glucocorticoids. As
M) led to a growth inhibition, whereas
16 INTERNATIONAL JOURNAL OF DERMATOLOGY January-February 1984 Vol. 23
TABLE 4. €ffect of Clucocorricoids on Proliferafron of Human Skin Fibroblasts
Clucocorticoid Concentration Cell Origin Response
Hydrocortisonelg lo- ' M Fetal normal + Hydrocortisone 10-7-10-5 M Hydrocortisone1 7-bufyrate Betamethasone- 1 7-valerate 10-8-10-s M Nicocortonide 10-n-10-6 M N icocortonide-2 1 -acetate 10-a-10-6 M
Prednisolone
- 10-7-10-5 M -
- - -
Hydrocortisone'" O.Ol-lpg/ml - 0.01-lpglml -
Hydrocortisone" 0.05-50 pg/rnl 0 Fluocinolone acetonide 0.05-50 pg/rnl + HydrocortisoneU 0.0001-10 pglml + Betarnethasonel 7-valerate 0.0001-10pg/ml + Clobetasone 17-butyrate 0.0001-10 pglml - Clobetasol-17-propionate 0.0001-10 pglml - HydrocortisoneU 10 pg/rnl 0 Clobetasone-17-butyrate 10 pglrnl -
Clobetasol- 1 7-propionate 10 pglrnl -
Betamethasone-1 7-valerate 10 pg/ml 0 HydrocortisoneePJ 10pg/rnl + Clobetasone-17-butyrate 10 pg/rnl - Clobetasol propionate 10 pg/rnl - Betarnethasone-1 7-valerate 10 pg/ml 0 Hydrocortisonez' 0.001 -20 pg/ml - Hydrocortisone-2 1 -acetate 0.001 -20 pglml - Hydrocortisone-17-butyrate 0.001 -20 pglml - Betamethasone-1 7-valerate 0.001 -20 Kglrnl - Clobetasol-17-propionate 0.001 -20 pg/rnl - Triamcinolone acetonide 0.001-20 pglml - Hydrocortisone= 10-z-l pg/ml Adult normal + Triamcinolone acetonide 10-3-1 pg/ml + Hydrocortisone= 50 pglml Normal adult + Hydrocortisone 50 pg/ml Keloid adult + Hydrocortisone" 0.3 pM Adult normal - Hydrocortisoneg 14 pM Fetal skin -
Hydrocortisone 14 pM Neonatal foreskin + Triamcinolone acetonide" 0-50 pgirnl Normal adult - Triamcinolone acetonide 0-50 pglml Keloid adult -
Non-genital
Embryonic normal
Fetal normal
Foreskin normal
Adult scleroderma
Adult forearm
Infant foreskin normal
Response: f . stimulation; -, inhibition; 0, no effect on the proliferation rate.
reported by Salomon et aI,* glucocorticoids suppressed the cellular elaboration of type IV collagenolytic activ- ity, whereas ECF stimulated the incorporation of amino acid into type IV collagen and in this way facilitated accumulation of the appropriate cell substratum for good growth of these cells and also of epidermal cells.3B
Salomon and Pramm found that growth-inhibitory ef- fects of glucocorticoids decreased with increasing con- centration of serum in the culture medium. Such culture conditions as frequency of medium renewal can also influence the final effect of glucocorticoids. The renewal
of glucocorticoid-containing medium leads to the disap- pearance of steroid-induced growth inhibition in many cases. We found3z that incubation of cells in the pres- ence of such concentrations higher than 1 pg/ml of one of the synthetic glucocorticoids, ie, clobetasol-17- propionate, led to a decrease of the cell number. How- ever, the proliferation rate returned to that of the control cells when the cells were supplied with fresh medium containing the same dose of glucocorticoid (Fig. 2 ) or even another glucocorticoid. This effect is known not to be due to either the appearance of steroid-resistant
No. 1 EFFECTS OF GLUCOCORTICOIDS Ponec 17
cells3' or a glucocorticoid-receptor deficiency.]] It i s possible that some postreceptor control function is al- tered. Similar effects were also found but not discussed by Ruhman and Berliner,'" Grasso and Johnson,32 and Salomon and Pratt.lo Recently, Strobel-Stevens and Lacey13 reported synthesis of a proliferation inhibitor by human cells in culture, which these cells release into the culture medium. Other authors report the presence of other mitotic inhibitors. It is conceivable that the synthe- sis of such inhibitors is influenced by glucocorticoids and that fibroblasts are not able to produce these in- hibitors after repeated application of glucocorticoids, which would explain the refractoriness of glucocorticoid-induced growth inhibition. From the examples given, it can be concluded that initial plating density, refeeding schedules, and the continuity and du- ration of application play an important role in the final effect of glucocorticoids on cultured skin fibroblasts.
The steroid-induced inhibition of cell proliferation (Table 4) was found to take place at concentrations seen in dermis in vivo after topical application of glucocor- ticoids ( 1 -1 0 P M ) . ~ Furthermore, good correlation be- tween the topical anti-inflammatory activity of various steroids and inhibition of cell growth has been reported by various authors.19.22~24,40,45.4fi Hollenberg4' showed that there is also good correlation between the degree of glucocorticoid-induced stimulation of amino acid uptake by human skin fibroblasts and the anti- inflammatory potency of these cells. Such effects could possibly be used for the screening of new anti- inflammatory steroids.
On the basis of many reports, it may be assumed that the changes in the cell-proliferation rate induced by glucocorticoids are mediated by a glucocorticoid recep-
The binding affinity of different steroids reflects the physiological potency as a inhibitor of the growth of a variety of Furthermore, in many cell systems the growth inhibition occurred in the same concentration range as the saturation of the binding sites of glucocor- ticoid-receptor. Our finding that glucocorticoid-induced inhibition of fibroblast proliferation takes place at steroid concentrations 50-1 00 times higher than needed for saturation of the receptor sites might be explained by accumulation of steroids in noncytoplasmatic compart- rnents of the cell. Furthermore, it has been shown re- peatedly'6 that glucocorticoid-induced growth inhibi- tion does not occur in glucocorticoid-resistant cells, where this effect i s associated with glucocorticoid- receptor deficiency. This resistance can be obtained by culturing cells for a relatively long time in the presence of a steadily increasing concentration of glucocorticoids. A diminished response to glucocorticoids in ageing cells was also accompained by a decrease in the number of glucocorticoid-receptor sites.'
At present, little information is available about the ef- fects of glucocorticoids on cultured human skin keratinocytes. There are a few reports on the role of hydrocortisone in the culture medium. Rheinwald and Green3 showed that in primary cultures of human keratinocytes, hydrocortisone had no effect on colony morphology, growth rate, or colony-forming capacity when a 3T3 feeder layer was present. However, in sec- ondary and subsequent cultures, where the cells are seeded at low density -lo-' cells/cm2), mor- phological changes occur, and the cell proliferation rate decreases in the absence of hydrocortisone. In the pres- ence of hydrocortisone, there was a two- to three fold increase in cell number in 2-week-old cultures.3 Since hydrocortisone has been shown to potentiate the effect of epidermal growth factor (by increasing the number of receptors for this factor) in other systems, it remains pos- sible that hydrocortisone affects proliferation by enhanc- ing the effect of the epidermal growth factor usually added to the culture medium. At hydrocortisone concen- trations higher than 10 pg/ml, Rheinwald and Green' observed suppression of epidermal growth. No hy- drocortisone or feeder layer is required when the seed- ing density in the primary culture is increased to 2.5 x lo5 cells per crnz.48 The presence of 5 x 10-jM hy- drocortisone was required for culturing primary epider- mal cells on fibronectin-coated dishes in serum-free medium at a density of 5 x 10' c e l l s / ~ m ~ . ~ ~ but not when at least 5 % fetal calf serum was added to the
8 -
7 -
6 -
1 -
1 L L 1 1 " '
1 2 3 L 5 6 7 8 9 - Recovety of cell proliferation either in the absence or in
the continued presence of high doses of clobetasol-17-propionate (C-17-P). C-17-P was added on day 1 at concentrations of 1 ( A), 2 (01, 4 (A), or 6 (B) pg/ml. C-17-P/ml was replaced on day 5 by fresh ster- oid-free medium (0) or by fresh medium containing 6 pg C-l7-P/ml (m). Steroid-free medium (01, medium with ethanol (XI . (For experi- mental data s e e reference 32.)
FIG. 3.
18 INTERNATIONAL JOURNAL OF DERMATOLOGY JanuaW-Februay 1984 Vol. 2 3
medium.j" Other investigators have reported similar findings. However, successful serial culture of human keratinocytes has been limited to a system where the teeder layer has been used and the culture medium con- tained hydrocortisone, a c-AMP stimulating agent, and epidermal growth iactor.j'
So far. no systematic study has been done on the ef- tects of the glucocorticoids used in dermatological prac- tice on the proliferation and differentiation of cultured human skin keratinocytes. Since the steroids can affect both the proliferation and differentiation of epidermal cells at the same time, suitable methods are required for simultaneous study of these effects in cultured human keratinocytes. Yasuno et al j2 found a slight decrease in epidermal growth and differentiation in organ cultures of adult human skin in the presence of glucocorticoids, and this effect led to prolonged survival of skin explants in vitro. In vivo experiments indicated that locally applied glucocorticoids have antimitotic activity that is corre- lated with therapeutic activity in humans and hairless- mouses4~5s cells. Voorhees et aljs reported approxi- mately 50% inhibition of the growth of epidermal cells of neonatal mice in the presence of 10-sM triam- cinolone acetonide. In contrast with these results, Lawr- ence and Christophers3' reported no effect of systemati- cally administered hydrocortisone on any phase of Swiss S mice epidermal-cell proliferation but an in- crease in the rate of epidermal differentiation that resulted in thinning of living epidermis. Similar obser- vations were made by Suzimoto et aIm in cultured em- bryonic skin, where differentiation was enhanced even at a very low hydrocortisone concentration (0.0001 Fglml), and this was accompanied by enhanced synthe- sis of some glycine-rich proteins. Delforno et a159 saw a reduction of epidermal thickness after topical applica- tion of various glucocorticoids. They found no signifi- cant reduction of the number of cells of viable epider- mis, but there was a reduction of cell size. Schwarz and Cottschling,GO who reported that locally applied triam- cinolone caused a selective increase of two serine- specific t-RNA in guinea pig epidermis, thought that this effect might be related to the process of differentiation of epidermal cells. Obinata and EndoGI reported induction of epidermal transglutaminase in cultured chick em- bryonic skin, which was accompanied by changes in the synthesis of epidermal structural proteins, insolubiliza- tion of proteins, and deposition of highly cornified cells.
Effects of Clucocorticoids on the Synthesis of Matrix Components
Effect on Collagen Synthesis
Collagen accounts for approximately 70% of the dry weight of skin. Human skin has two main collagens, type
I (about 80%) and type Ill [about 20%).62 In skin atrophy caused by topically applied glucocorticoids, the dermis shows considerable thinning and loss of collagen. This seems to suggest eitects of glucocorticoids on collagen hiosynthesis or degradation.
Briefly, procollagen (the collagen precursor) i s syn- thesized in the cells and after its release, the conversion into collagen takes place outside the cell. Collagen con- tains unique amino acids, ie, hydroxyproline and hy- droxylysine, which are necessary for its stability and maturation. The synthesis of these amino acids occurs post-translationally, and is catalyzed by prolyl and lysyl hydroxylases, enzymes that require a-keto-glutarate, Fe2+, ascorbic acid, and 0, as cofactors. The level of prolyl hydroxylase in the tissue has been considered to control the rate of collagen synthesis. The final structure of collagen fibers may be affected by other post- translational modifications, which are dependent on the activity of other enzymes, such as galactosyl and glucosyl transferase or lysyl oxidase, and the activity of these enzymes may also affect the rate of collagen syn- thesis. The process of collagen formation might also be influenced by the activity of enzymes that catalyse the conversion of procollagen into collagen (for review, see'). All of these steps as well as the rate of collagen degradation may be influenced by glucocorticoids.
The effects of glucocorticoids on collagen biosyn- thesis are summarized in Table 5. These effects can occur either with or without a concomitant effect on the total protein synthesis. Only in one caseno concerning fetal human skin fibroblasts has an increase in both col- lagen and noncollagenous protein synthesis been re- ported. In all other studies, either a decrease or no effect on collagen synthesis was found. The decrease of colla- gen synthesis was usually greater than that seen for non- collagen synthesis. In some other studies done in soil- ated fibroblasts, glucocorticoids decreased collagen synthesis, whereas of noncollagenous protein synthesis was
Results concerning the effects of glucocorticoids on collagen synthesis in cultured human skin fibroblasts are less controversial than those concerning the effects on cell proliferation. The diversity of the cell populations used in different laboratories i s probably less important for studies on collagen synthesis. This i s indicated by the recent studies done by Tajima and Pinell,sl who showed that fibroblasts originating from papillary and reticular dermis differed as to growth characteristics but were very similar with respect to collagen synthesis. Some systems have been reported to show a decrease in cellu- lar prolyl hydroxylase activity concomitant with the de- crease in collagen synthesis. Recently, Rokowski et als' reported that steroid treatment of rats resulted in a de- crease of functioning polysomal messenger RNA for col-
Met
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No. 1 EFFECTS OF GLUCOCORTICOIDS Ponec 21
lagen, but they did not observe a similar decrease for prolyl hydroxylase messenger RNA. The decrease in pro- lyl hydroxylase activity seen in some studies might be due to the presence of an inactive protein that i s a smal- ler subunit of the tetrameric enzyme. The activity of the enzyme catalyzing the synthesis of tetrameric enzyme from subunits might be influenced by glucocorticoids such that the prolyl hydroxylase activity would be de- creased. However, the overall degree of hydroxylation of the collagen synthesized in the presence of glucocor- ticoids was found to be uninfluenced by decreased pro- lyl hydroxylase acti~ity."- '~-'~ Murad et als2 recently found no correlation between prolyl hydroxylase activity and collagen production, which means that the rate of collagen synthesis i s not controlled by the level of prolyl hydroxylase. In addition to the effect of glucocorticoids on prolyl hydroxylase activity, two of these studies showed a decrease in the activity of lysyl hydroxylase and also of galactosyl and glucosyl transferase activ- ity.77.7Y The latter two enzymes are involved in the syn- thesis of hydroxylysyl-bound mono- en disaccharides.
The conversion of procollagen into collagen occurs extracellularly, two or more proteases (procollagen- peptidase) cleaving the carboxy- and amino-terminal residues. N-terminal procollagen peptides were reported to exert feedback control of collagen synthesis.' Nothing is known about any effect of glucocorticoids on the con- version of procollagen into collagen or steroid-induced changes of the ratio between type I and type Ill collagen, or about the activity of lysyl oxidase, an enzyme in- volved in the formation of cross-links in collagen fibers. The turnover of collagen i s determined not only by the rate of collagen synthesis but also by the activity of t is- sue collagenase, which catalyses collagen degradation. Glucocorticoids have been shown to have an effect on collagenase activity. Koob et al . R S described a glucocorticoid-induced reduction of enzyme activity in various in vitro systems, and Hook et alR4 found stimula- tion of such activity in corneal fibroblasts. Recently, McCoy et aIz9 found that degradation of collagen was enhanced by triamcinolone acetonide in cultures of human skin fibroblasts. In most of the other studies in which the experiments were performed in the presence of serum, a selective decrease of collagen synthesis was probably not accompanied by collagen degradation, be- cause serum is known to contain inhibitors of this enzyme.
These studies indicate that glucocorticoids can simul- taneously enhance collagen degradation and selectively inhibit collagen synthesis by fibroblasts in culture.
As was the case for effects of glucocorticoids on cell proliferation, there was good correlation between the steroid-induced inhibition of collagen synthesis in fibro- blasts and the anti-inflammatory potency of the steroids.
Effect on the Synthesis of Clycoarninoglycans
Next to collagen, the glycoaminoglycans form the second most important matrix component. The glucocorticoids were found to influence the synthesis of various glycoaminoglycans in vivo and in v i t r ~ . ~ ~ - ~ ~ In vitro studies with cultured human skin fibroblasts showed that the synthesis of various glycoaminoglycans responds differently to glucocorticoids. Saarni and H o p s u - H a ~ u ~ ~ ~ ~ ~ found that the concentration of steroids necessary to inhibit the synthesis of hyaluronic acid was about lo4 times lower (at steroid concentrations between 1 O-" and 1 0-8M) than the concentration needed to in- hibit the synthesis of sulfated glycoaminoglycans and about 1 O5 times lower than was required for the inhibi- tion of the collagen synthesis. Furthermore, the effects of steroids on the synthesis of hyaluronic acid were also more pronounced, even at low concentrations of the steroid, than on that of sulfated glycoaminoglycans. Ac- cording to Priestley et at,22 glucocorticoid-induced in- hibition of the synthesis of sulfated glycoaminoglycans took place at lower steroid concentrations that were re- quired for the inhibition of cell proliferation.
The effect of glucocorticoids on the synthesis of sul- fated glycoaminoglycans was found to be time depen- dent. Short incubation (1 2 and 24 hr) caused a decrease, but the prolonged treatment (48 hr) did not affect the synthesis of glycoaminoglycans.88
The early effect of steroids in low concentrations on the synthesis of hyaluronic acid may trigger the early structural changes is connective tissue. Since hyaluronic acid has been reported to be involved in the regulation of the diffusion of water, salts, nutrients, and mac- romolecules between the circulation and cells, the glucocorticoid-induced changes of hyaluronic acid syn- thesis might affect cell rnetabol i~m.~~ Cell surface hepa- ran sulfate too was recently reportedsg to be involved in the regulation of cell proliferation, since an enrichment of cell surface by heparan sulfate in late passage of human diploid fibroblasts, was proportional to the de- crease in saturation density. Treatment of cells with heparitinase completely abolished the inhibitory effect. Glucocorticoid-induced changes in glycoaminoglycan synthesis can also indirectly affect cell proliferation.
As found for the effects of glucocorticoids on collagen synthesis and cell proliferation, the degree of inhibition of synthesis of hyaluronic acid by various glucocor- ticoids is correlated with their clinical potency.86
Glucocorticoids and Prostaglandins
Since prostaglandins were reported to regulate the in- flammatory response, part of the anti-inflammatory ac-
INTERNATIONAL IOURNAL OF DERMATOLOGY lanuary-February 1984 Vol. 23 22
tion of glucocorticoids may be due to their effect on the prostaglandin system.
Glucocurticoids have been shown to reduce prosta- glandin synthesis in several r e l l types.li.nH*Yu-y’ In cultured embryonic mouse fibroblasts, Peters et al’” found a dose-dependent suppression of prostaglandin production by prednisolone and also saw suppression of the cyclic AMP content and synthesis of glycoaminogly- cans in these steroid-treated fibroblasts. The steroids even suppressed the prostaglandin-stimulated accumu- lation of cyclic AMP and the increased synthesis of glycoaminoglycans. These findings suggest that glucocorticoids can influence cellular metabolism in normal as well as in inflamrned tissue, where the level of prostaglandins is elevated. The decreased prostaglandin production was due mainly to the decrease of prosta- glandin release.
HammertGm et alw showed that in involved psoriatic epidermis, the increased level of arachidonic acid and 12L-hydroxy-5,8,10,14 eicosatetraenoic acid (HETE) was normalized after the topical application of glucocor- ticoids (0.05% diflorasone diacetate). These authors offer a possible hypothesis on the effects of glucocor- ticoids on the phospholipase activity. The hypothesis was later confirmed by Johnson et al,” who found that, in cultured endothelial cells, the glucocorticoid-induced inhibition of prostaglandins production was attributed to the induction of the synthesis of an inhibitor of phospholipase.
In many cases, good correlation has been found be- tween the binding of glucocorticoids to the receptor, the effects on the proliferation rate and on the synthesis of matrix components, and the anti-inflammatory potency of these steroids. This suggests that the models provided by cultured human skin fibroblasts and keratinocytes are suitable for studies on the biochemical events that take place after the administration of glucocorticoids.
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President Wilson: Influenza and Encephalopathy
Weinstein originally postulated that Wilson had another stroke on April 3, 1919, during the Paris Peace Conference. Later he changed his diagnosis to "virus encephalopathy, which, superimposed on preexisting brain damage, produced changes in behavior which may have influenced the outcome of the peace negotiations." We have a degree of medical documentation for this period, since Wilson's personal physician, Cay Grayson, kept a diary during the conference. Wilson was apparently in good health through March, except for minor ailments. He told Grayson at one point, "It is true I had a headache, neuralgia, sore-throat, tooth-ache, fever and a chill, and my equatorial zone has been on a strike. . . . I am suffering from a retention of gases generated by the Republican Senators-and that's enough to poison any man." Wilson was under considerable pressure to meet British, French, and Italian demands to impose peace terms that were incompatible with his Fourteen Points and to seek certain amendments to the Covenant of the League of Nations to quell opposition at home. The conference was at its climax when he fell ill on the afternoon of April 3.
Wilson complained of "intense pains in my back and stomach and head" and "a very severe coughing spell." Grayson found that his temperature was 39°C (103"F), diagnosed influenza, and promptly sent him to bed. The next morning his condition was distinctly worse, and word was sent to Prime Minister Lloyd George that Wilson could not be present at the morning session. However, by April 5 Wilson's temperature was down to 38°C ( lO l "F) , and he began to conduct conference business from his bedside. He met at his house with other members of the Big Four on April 8 and resumed a full conference schedule on April 9. Throughout this period, Mrs. Wilson entertained luncheon guests and made goodwill visits in Paris; this suggests that there was no serious alarm about Wilson's condition.-Marrnor MF: Wilson, strokes, and zebras. N Engl I Med 307:528, 7982