Interleukin-8 Stimulates Calcium Transients and Promotes Epidermal Cell Proliferation

Andrea Tuschil, Charles Lam, Alexander Haslberger, and Ivan Lindley Sandoz Forschungsinstitut, Vienna, Austria

The presence of large amounts of biologically active inter­ and concentration dependent. Half maximal effect was ob­ leukin-8 (IL-8) in psoriatic involved skin suggests that it may served at 1.2 nM. Normal also responded to contribute, in part, to the changes observed in , in­ IL-8 (6 nM) by rises in cytosolic free Ca++ from a pre-stimu­ cluding hyperproliferation ofkeratinocytes. To examine the lated level of 269 nM to transient peak value of 393 nM. In effect of IL-8 on epidermal growth, we monitored cytosolic addition, IL-8 promoted epidermal cell proliferation. P?ly­ free Ca++ transients in human keratinocytes adult skin epi­ clonal anti - IL-8 antibody blocked IL-8 - induced calciUm dermis calcium reduced level, temperature elevated (HaCat) changes and proliferation. Under similar conditions, human cells and normal keratinocytes loaded with the cell perme­ also responded to IL-8 in a similar dose range by.a able, acetoxymethyl derivative, indo-lAM. Addition ofIL-8 rapid and transient mobilization of Ca++. The findings indi­ (0.06 - 47 nM) to the HaCat cells induced rapid rises in cyto­ cate that IL-8 has a wider range of responsive target cells than solie free Ca++ from resting levels of 145 ± 38 to peak levels hitherto thought and acts as an autocrine . of 889 ± 10 nM. The induced rises in Ca++ were transient ] Invest Dermato199:294-298, 1992

soriasis is a disease characterized by hyperproliferation of cules at the inflamed site (reviewed in [3]). Among potential proi~­ keratinocytes, and accumulation of activated T lympho­ flammatory that are detectable in psoriatic skin in biolog~­ cytes and neutrophils in the dermis and epidermis [1- 3]. cally active form are members of a family of small chemotactic The molecular basis for the hyperplasia ofkeratinocytes peptides, of which -8 (IL-8) and monocyte chemotactic or the increased trafficking of activated T cells and neu­ factor (MCF) have been increasingly studied [4 - 6]. Although ongl- trophilsP into the skin appears to depend on locally produced growth nally identified by its effects on neutrophils, IL-8 is now known to factors and chemoattractants and the expression of adhesion mole- stimulate ofT cells [7] and basophils [8], and to induc release in basophils [9] . IL-8 is produced by various ce ilI types including lymphocytes, monocytes, neutrophils, endothelia cells, dermal fibroblasts, and keratinocytes in response to inflamma­ tory mediators such as interleukin-1 (IL-1), tumor necrosis fac~r Manuscript received May to, 1991; accepted for publication March 9, alpha (TNF-a), gamma (IFN-y), or lipopolysacchan ~ 1992. (LPS) [10 ,11 ]. Intradermal or intracutaneous administration ofIL"4J Reprint requests to: Andrea Tuschil, Sandoz Forschungsinstitut, Brunner into experimental animals [7,11,12]' or into human skin [13,1 Strasse 59, A-1235 Vienna, Austria. causes an characterized by the accumulation of neu- Abbreviations: [Ca++li: cytosolic free calcium trophils and lymphocytes at the sites of injection. . DMEM H21: Dulbecco's modified minimal essential medium In view of the indigenous cellular producers of IL-8 and t~elr EGF: epidermal growth factor different responsiveness to proinflammatory cytokines [10,11], ItlS EGTA: ethylene glycol tetraacetic acid possible that IL-8 and its inducing mediators could be involved In FCS: fetal calf serum the recruitment and proliferation of epithelial cells in cutaneouS FMLP: formylmethionyl-Ieucyl-phenylalanine inflammation. The observation that IL-1a, TGF-a, and IL-6 are HaCat: human keratinocytes adult skin epidermis calcium reduced present in psoriatic skin [15 - 17] and that some cytokines can pro­ level, temperature elevated mote proliferation of keratinocytes in cultures [18 ,19] indicate that HBSS: Hanks' balanced salt solution cytokines could potentially contribute to the profound kerati~ocytd IFN-y: IgG: immunoglobulin G hyperplasia seen in psoriasis. Indeed, recent studies have imph~ater_ IL-l: interleukin-l IL-8 as both a potent chemoattractant [20] and a mitogen for eplde IL-6: interleukin-6 mal keratinocytes [21 ,22]. . One indicator of responsiveness of eukaryotic cells to certailf IL-8: interleukin-8 0 KBM: basal medium growth factors is a transient rise in the intracellular concentratl0 KGM: keratinocyte growth medium free calcium ions ([Ca++]i) [23]. In this study, we examined wheth er LPS: lipopolysaccharide or not HaCat cell line and normal human foreskin keratinocytes, MCF: monocyte chemotactic factor like human neutrophils, respond to IL-8 by mobilization of cyto~O~ PBS def.: phosphate-buffered sa line without calcium and magnesium lic free calcium ([Ca++]i)' We also investigated whether any IL- 1 IOn SDS: sodium dodecyl sulphate induced rise in [Ca++]i was associated with stimulation of epldermad TCA: trichloroacetic acid cell proliferation as measured by [3H]thymidine incorporation ~n TGF-a: transforming growth factor alpha increase in cell numbers. The results show that under appropnatd TNF-a: alpha experimental conditions, IL-8 raises [Ca++]i in HaCat ceJls an

0022-202X/92/S05.00 Copyright © 1992 by The Society for Investigative Dermatology, Inc.

294 VOL. 99, NO. 3 SEPTEMBER 1992 C.++ CHANGES AND MITOGENIC EFFECT OF IL-8 295 human foreskin keratinocytes and promotes their proliferation, suggesting that this could contribute to the keratinocyte hyperplasia seen in psoriatic lesions. MATERIALS AND METHODS Source ofIL-8 Recombinant human IL-8 was purified to homo­ geneity from Escherichia coli expressing the synthetic IL-8 as ... ~.~--. previously described [24]. Stock solutions of IL-8 were prepared in IL-8 phosphate-buffered saline (PBS) containing 0.25% bovine serum f·MLP .!! albumin as a carrier and stored at - 20 ° C. Specific poly­ 366nM fUCat - clonal antibody to IL-8 was raised in rabbits and immunosorbent 178nM f punfied as previously described [25]. No endotoxin contamination - ~ (s 12.5 pg endotoxin activity Img protein) was detected with the IL-8 bradykinin ~ chromogenic Limulus amebocyte lysate reagent kit (Wittacker, MA, Bioproducts, Inc, Walkersville, MD) in either IL-8 or immun­ osorbent purified antibody preparations. -393nM 269nM Cultivation of HaCat Cell Line The HaCat cells were main­ - tained by subculturing in endotoxin-free modified Dulbecco's min­ IL-8 anal essential mediu~ (DMEM H21) enriched with 5% fetal calf bradYkinin serum (FCS) and 2.5% glutamine on plastic cover slips in Leighton tubes at 3rc in a humidified incubator [26]. At about 80% con­ < 1 mla·ute > fluency, the DMEM H21 was replaced with endotoxin-free kera­ tlnocyte basal medium (KBM) (Clonetics) containing 0.06 mM Figure 1. Stimulation of cytosolic free calcium transients in ad herent Ca++, and the cells were cultured for 2 d to induce quiescence as HaCat cells, normal foreskin keratinocytes, and suspended human blood neutrophils by IL-8 (6 nM), bradykinin (2 JiM), or FMLP (0.2 JiM) . Indo- previously described [27]. 1-loaded neutrophils and epidermal cells were exposed to the indicated Cultivation of Normal Human Epidermal Keratinocytes stimuli, followed 2 - 3 min later by a second dose of FMLP or bradykinin, Normal human keratinocytes were obtained from fresh neonatal respectively. Cytosolic calcium was measured as described in Materials atld Methods. Arrows, times of addition of the stimuli. f?reskin biopsies. Epidermal cells were disaggregated by trypsiniza­ tl~n overnight at 4 ° C in Hanks' balanced salt solution (HBSS) Without Ca++ and Mg++ containing 0.25% trypsin. The cells were plated out at 107 cells per 50 ml culture flask in endotoxin-free ~eratinocyte growth medium (KGM) containing 1.0 mM Ca++ and def. and 200,111 of ice-cold 10% trichloroacetic acid (TCA) was

In~ubated at 37°C under 10% CO2 , Normal keratinocytes used in added, and the TCA-precipitated nucleic acid solubilized in 100,111 thiS study were derived from the second to fourth passages grown as of 0.1 M sodium hydroxide, 1 % sodium dodecyl sulphate (SDS) and a rnonolayer to 80% confluency on cover slips. The monolayers 2% sodium carbonate for 30 min and counted in 2 ml Optifluor Were then transferred to KBM containing 0.06 mM CaCl2 and (Packard). starved for 48 h at 37°C as described above for the HaCat cells. Isolation of Human Neutrophils Neutrophils were prepared Ass~y of Cytosolic Free Ca++ in Keratinocytes The KBM from fresh blood obtained from healthy volunteers. The cells were ill.e~lUm was replaced with a loading medium, i.e., fresh KBM con­ purified by centrifugation through a layer of Ficoll-Paque and sedi­ talnmg 2,uM acetoxymethyl ester of indo-1 (indo-l1AM). The mentation through dextran followed by hypotonic lysis of residual Leighton tubes were immersed in a 3rc bath and rotated so that erythrocytes [29]. The isolated preparation contained the coverslips were flopped gently back and forth through the load­ > 98% neutrophils, and cell viability was always 2: 95% as deter­ ~ng me.dium. After incubating for 30 min, coverslips were t~ans­ mined by trypan blue exclusion. erred lUto fresh KBM and then stored in the dark for 15 mm at For the measurement of Ca++, the neutrophils were incubated roorn temperature. The loaded HaCat cells were viable as judged by with 2,uM acetoxymethyl ester of indo-l (indo-l I AM) in HBSS trypan blue exclusion (2: 95% viability). without Ca++ and Mg++ for 30 min at 37°C. The loaded cells were C ~~e coverslips were rinsed once in HBSS containing 0.06 mM washed once and suspended in HBSS containing 1 mM Ca++. Cy­ a ,placed in standard l-cm square quartz cuvettes contammg the tosolic free Ca++ levels were measured as described above but in sarne buffer, and transferred to a Perkin-Elmer fluorescence spectr.o­ suspension cultures following addition ofIL-8 at various concentra­ ph Otometer (model LS-5B) maintained at 37°C with constant stu­ tions, FMLP or the PBS containing bovine serum albumin. Fmn and ring. After equilibration for 5 min, IL-8 at various concentrations, Fmin were recorded after the addition of 0.05% Triton X-I00 and or the PBS containing the carrier protein, was added and the result­ ethylene glycol tetraacetic acid (EGTA) at pH 8.4, respectively. ~g fluorescence recorded (excitation, 330 nm; emission, 400 nm). Statistical Analysis Differences between groups were deter­ t the end of each assay, a calibration to determine F max and F min was mined by a Student t test. performed by adding ionomycin (5 ,11M) and MnCl2 (1 mM), respec­ ~vely . The Ca++ levels were calculated according to the method of RESULTS rynktewicz [28]. Stimulation of Calcium Transients in Keratinocyte-Like ~eratinocyte Proliferation Assay Keratinocytes were plated at HaCat Cells and Human Blood Neutrophils by IL-S As I< >< 103 cells per well of a 96-well microtiter plate and cultured in shown in Fig 1, the addition of IL-8 at a standard concentration of w~M or KBM for 3 d. The medium in each well was then replaced 24 nM induced a rapid and transient rise in cytosolic free Ca++ in £Ith KGM or KBM supplemented with epidermal growth factor adherent indo-1-loaded quiescent HaCat cells, from a resting level ( GF) (0.01 ,ug/ml), IL-8 at various concentrations, or the PBS of 145 ± 38 nM to transient peak values in excess of 800 nM. The Contaming the carrier protein, and after 4 d replaced again with the induced rises in [Ca++]i were dose-dependent and peak values were ~~e s upplemente~ medium for a further 2~ h. Proliferatio~ ~as observed at IL-8 concentrations of about 20 nM (Fig 2). Trypsiniza­ ( aS ~red by labelmg cultures with 0.5 ,uCI ml- I [3H]thymldme tion of the HaCat mono layers for use as suspension cultures abol­ thPeclfic activity 25 Ci mmol- I , Amersham International) during ished the calcium transients stimulated by IL-S. It is not clear at ill. e last 24 h of incubation. After labeling, the medium was re- present whether this effect is due to trypsin sensitivity of the IL-8 oved, each well was washed 3 times with 200,111 of ice-cold PBS receptors on the HaCat cells. Evidence that the induced rises were 296 TUSCHIL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

1000 r------~ rapid. Again, IL-S - induced Ca++ transients in neutrophils were 950 dose-dependent (Fig 2) and could be inhibited by excess neutraliz­ 900~ ing rabbit polyclonal anti-IL-8 (data not presented). 850 Characteristics of the Response of HaC at Cells to IL-8 The 800 response of the HaCat cells to IL-S was transient, i.e., cytosolic f:ee 750 1 Ca++ rose to peak levels and gradually fell over the next few mlO­ 700i, utes (Fig 1). A second addition ofIL-8 had no further effect on the ~ 650! levels of cytosolic free Ca++, i.e., the first addition oflL-S rendered c:: the cells non-responsive to a second application. Incubation of the ...... HaCat cells with pertussis toxin (l,Ltg/ml) for 1.25 h at 37°C abol­ + ished the responsiveness of the cells to IL-S. Whereas [Ca++]i ro~e +CO from resting levels of 211 ± 47 nM to peak levels of 439 ± 103 10 U...... control cells stimulated with IL-S at 2.5 nM, pertussis toxin-treated cells stimulated with a similar concentration of IL-8 showed nO calcium transients. Thus, the resting levels of [Ca++]i of 227. ± 4 nM in pertussis toxin-treated cells remained more or less unlO­ fluenced at 17S ± 93 nM following stimulation with IL-8. at 2.5 nM. These data indicate that guanosine triphosphate binding (G-proteins) are involved in transducing the stimulatory signal of IL-S. Lastly, IL-S did not cause any detectable rise 10 cytosolic free Ca++ in the absence of extracellular Ca++ (data not 9.5 24 46 presented). [nM] Responses of Normal Keratinocytes to IL-8 In Vitro !he 2100r------addition of nanogram quantities of IL-S also stimulated calcium 2000 transients in adherent normal keratinocytes (Fig 1). However, com­ 1900 (... 01 8) pared to HaCat cells, normal keratinocytes responded weakly to 18001 IL-S stimulatory effects. For example, in response to 6 nM IL-S, 17001 cytosolic calcium level increased from a resting level of 269 nM 1600) to transient peak value of only 393 nM in riormal keratinocyt~S, 1500 whereas transient peak values in excess of SOO nM were recorded In . ~ 1400 HaCat cells stimulated with similar concentrations of IL-S. T~e c:: 1300 normal keratinocytes also showed significant variability in their e ;=, 1200 responses to the cytokine. Nevertheless, whenever the cells we-: tl +1100 ! responsive to IL-S, the addition of excess neutralizing rabbit an .­ CO 10001 IL-S polyclonal antibody blocked Ca++ transients. Normal rabbit ~ 900' serum immunoglobulin G (IgG) preparation used as a negative con­ 800 1 trol did not influence calcium transients in keratinocytes (data not 700f presented). 600i 5001 Mitogenic Effect ofIL-8 for Normal Keratinocytes Th~ I?i­ to genic effect of IL-S was assessed by measuring 400: PH]thymldlO~ incorporation into TCA-precipitable counts. The results in Table show that normal keratinocytes did not proliferate significa~t1y 10 ICBM alone as assessed by the small amounts of[3H]thymidine mcor- po rated by control cultures. The addition of increasing concentra­ o .3 .6 1.2 2.8 6 1214 tions of IL-8, however, resulted in three- to sevenfold increase III INTERLEUKIN-8 [nM]

Figure 2. Comparative stimulation of cytosolic free Ca++ transients in indo-l -loaded HaCat cells (A) and blood neutrophils (B) by IL-8. Indo-l­ Table I. Interleukin-S Stimulates Proliferation of Normal loaded cells in duplicates were stimulated by IL-8 and cytosolic calcium was Foreskin Keratinocytes' ____ measured. Neutrophils were isolated from three different donors for every [3H] Thymidine Incorporation experiment. Results are the mean ± SD of more than six experiments. (CPM ± SD) __ an effect of IL-S rather than contaminants was provided by the Stimulus Concentration use of immunosorbent-purified anti - IL-S polyclonal antibody. (nM) KBM KGM ------Whereas HaCat cells responded to IL-S at 24 nM by raising resting 51.7 ± 40 13,827 ± 3,810 calcium levels from lSS ± 23 nM to peak values in excess of IL-8 0.012 181.3 ± 46 15878 ± 737 1050 nM, no calcium transients were observed when IL-S at 24 nM 0.06 164.7 ± 44 17: 106 ± 1,98!b 6 was pre-incubated with excess neutralizing polyclonal anti-IL-S 0.12 149.3±165b 19549±9,5 antibody (2 ,Ltg/ml). For example, resting level of[Ca++]i in HaCat 1.2 175.3 ± 34 15'798 ± 2,908 6.0 163.3 ± 127 16'408 ± 1,685 cells measured in the presence of the antibody was 201 ± 12 and in 9 cells stimulated with IL-S pre-incubated with the antibody was 12.0 384.0 ± 360 16'545 ± 3,11 1S3 nM. Normal rabbit serum IgG preparation did not influence EGF 0.016 1273.0 ± 846 15' 506 ± 3,888 ' 139 ± 1,598 either the resting or stimulated level of [Ca++]i in HaCat ______0_ .0_8_1 ______7_ 0_7_.7_±__ 4_ 4_2 ______10,------cells. • Keratinocytes were cultured for 3 d, the indicated medium supplemented w~~ either IL-8 or EGF added and cells were cultivated for a further 5 d. Proliferation olts Under similar experimental conditions, IL-S or FMLP stimulated s rapid rises in cytosolic free Ca++ in neutrophils (Figs 1 and 2) to estimated by ['HJthymidine incorporation during the last 24 h of culture. The r. cWo peak values of about 2000 nM. Also, whereas the fall of peak cyto­ (CPM ± SD) are representative of three separate experiments that used cells froIll different donors. CPM, counts per minute; SD, standard deviation. trol solic free Ca++ to resting levels in HaCat cells was protracted, the b Addition of immunosorbent purified anti _ IL-8 antibody reduced counts to con fall of calcium to resting levels in the neutrophils was relatively levels, whereas addition of normal rabbit serum IgG had no effect. VOL. 99, NO.3 SEPTEMBER 1992 Ca++ CHANGES AND MITOGENIC EFFECT OF IL-8 297

Table II. Effect of Interleukin-S on the Proliferation of HaCat and activation of inflammatory cells, the role ofIL-S as an autocrine Cells In Vitro" or paracrine growth factor regulating the characteristic keratinocyte hyperproliferation seen in skin diseases such as psoriasis is as yet [lH]Thymidine Incorporation unclear. (CPM ±SD)' The results presented here demonstrate that IL-S raises the Stimulus Concentration [Ca++]; of keratinocyte-like HaCat cells and normal human fore­ (nM) KBM KGM skin keratinocytes adhering to a substratum. It is not clear whether most of the Ca++ mobilized in HaCat cells in response to IL-S came 12,845.3 ± 3,001 88.7 ± 16.8 from an influx of exogenous Ca++, and/or from intracellularly IL-8 0.012 117.3 ± 17.5 13,122.0 ± 728 0.12 137.7 ± 35.5' 15,060.7 ± 1,101' stored Ca++. Whatever the source, the IL-S-stimulated rise in 1.2 129.7 ± 13.8 12,663.7 ± 2,589 [Ca++]; was transient, peaked within 30 seconds, and gradually de­ 12.0 218.0 ± 19.5 11,965.0 ± 740 clined to basal levels within 1 to 2 min. Once the [Ca++]; returned to EGF 0.016 1,497.0 ± 207 11,903.0 ± 372 basal levels, a second addition of the cytokine had no effect (data not presented), but it did not interfere with a further rise in [Ca++]; • Experiments were performed as in Table I using HaCat cells plated out at 5 X 10' per well. Representative data of three experiments arc shown. when an heterologous agonist such as bradykinin was added. The • CPM, counts per minute; SO, standard deviation. peak levels of the mobilized [Ca++]; were dose dependent. The , Immunosorbent purified anti - IL-8 reduced counts to controllevcls whereas nor­ stimulation of human keratinocytes by IL-S was lower and variable tnal rabbit IgG had no effect. in samples obtained from different volunteers. This might be the reason why the effect of IL-S on keratinocytes has been demon­ strated only in some studies [20-22]. It is also possible that differ­ ences in the responsiveness of HaC at and normal keratinocytes were the amounts of [3H]thymidine incorporated by the keratinocytes. due to culture conditions to which the cell preparations were ex­ Under similar conditions, EGF increased proliferation by up to 26 posed 4S h prior to analysis. To avoid stratification and detachment tlrues. Evidence to support that IL-S - induced proliferation was due from the slide, normal keratinocytes could be grown only in KGM, to the mitogenic effect of the cytokine rather than to contaminants a medium with a lower calcium concentration than the recom­ In the preparation was provided by the use of immunosorbent puri­ mended medium (DMEM) for cell growth [32]. Evidence that the fied neutralizing polyclonal anti - IL-S. Whereas normal rabbit calcium responses observed here were not due to any active contami­ serum IgG preparation had no effect on IL-S stimulated prolifera- nants was provided by neutralization of the cytokine with specific 11on, anti - IL-S antibody reduced proliferation rate to background immunosorbent-purified anti - IL-S antibody. Pre-incubation of the evels as evidenced by (3H]thymidine incorporation. In KGM alone, IL-S with excess neutralizing polyclonal anti-IL-S rendered the the proliferation rate of keratinocytes was high. Nevertheless, IL-S preparation ineffective in eliciting the calcium response in the also enhanced the proliferation by 10% to 40%, levels comparable HaCat cells, whereas pre-incubation of the cytokine with normal to that observed with EGF. rabbit serum IgG did not interfere with the stimulatory effect of The mitogenic effects ofIL-S on HaCat cells were also examined IL-S. (Table II) . IL-S increased [3H]thymidine incorporation by HaCat Two lines of evidence suggest that stimulation of the keratino­ cel1s cultivated in KBM dose dependently. Again, purified normal cytes by IL-S was induced through receptor-mediated events. rabbit serum IgG had no effect on IL-S-stimulated (3H]thymidine Firstly, pre-treatment of the HaCat cells with Bordotella pertussis InCorporation, whereas polyclonal anti - IL-S antibody reduced the toxin for 1.25 h abrogated the calcium response, suggesting that cytokine-mediated effects. A slight mitogenic effect was also ob­ IL-S stimulated calcium transients through a pathway involving G served when the cells were cultivated in KGM. The effect, how­ proteins [33]. Secondly, trypsinization of the adherent cells for use ever, was not dose dependent. Under similar conditions, EGF at 0.1 in suspension cultures abolished the calcium transients. Under simi­ ng/ml also did not promote HaCat cell proliferation as was ob­ lar experimental conditions, specific receptors for IL-S on the served in KBM. HaCat cells could not be detected (J. Besemer, personal communica­ In. several experiments, the mitogenic effect of IL-S was also tion). We cannot at present exclude the possibility that the lack of rOnltored by direct microscopic determination of cell numbers. specific binding of IL-S to epidermal keratinocytes in suspension L-S at 0.12 nM increased HaCat cell counts by 54.S% ± 23.2% cultures was due to the low numbers or to the sensitivity of the oVer the counts obtained in control cultures. Again, polyclonal receptors to trypsin. Nevertheless, the observed desensitization of anti - IL-S antibody reduced cell counts to control levels. Compared the cells to second applications of IL-S could be explained by the ~o the enhancement of (3H]thymidine incorporation of 55.2% ± presence of IL-S receptors on keratinocytes. 0% at IL-S of 0.12 nM, the effect of IL-S was similar for both Recent preliminary reports show that IL-S is a chemoattractant assays. These findings indicate that at the cell density used here for HaCat cells [20] and promotes the proliferation of normal kerati­ catabolism of thymidine did not significantly result in underestima­ nocytes in vitro [21,22J. The results presented here confirm the tion of the mitogenic effect of IL-S. mitogenic effect of IL-S for HaCat cells and extend these reports by showing that normal human foreskin keratinocytes also respond to DISCUSSION IL-S by mobilization of [Ca++]; and by enhanced proliferation. The ll-S is now thought to play an important role in eliciting the neu­ proliferation was best detected after 5 d incubation in KBM. As the tr[phil and the lymphocyte infiltrates that are characteristic features level of free calcium in the skin is reported to be low [34], we ~ early developing and established psoriatic lesions [5 - 7,30,31]. speculate that locally produced IL-S could promote, at least in part, Upport for the potential role ofIL-S in the pathogenesis of inflam­ the hyperproliferation of epidermal cells in certain cutaneous in­ matory skin diseases include its potent proinflammatory effects flammatory diseases. Evidence supporting this contention come w[l hen injected in experimental animals [9,11,12] and volunteers from reports showing that Ca++ regulates many aspects of epider­ 3.,14], and the detection of considerable amounts of biologically mal differentiation [35-37]. Keratinocytes grown in a medium ~Ctlve IL-S and related peptides in psoriatic epidermis [5 - 7,30,31]. containing $ 0.1 mM Ca++ are phenotypically similar to basal epi­ dn addition, the presence of the cytokine in other inflammatory dermal cells, whereas cells grown in media containing high Ca++ Iseases, such as rheumatoid arthritis [25,32]' and septic shock [10] (~ 0.1 mM) have many aspects of suprabasal phenotype in vivo. at /evels reflecting disease activity indicates a possible pathogenic Taken together, the results suggest that IL-S has a wider range of fib e. Furthermore, cells indigenous to the inflamed sites, including target cells than hitherto thought. v' roblasts, keratinocytes, and endothelial cells, can be stimulated in Although the precise mechanism{s) by which Ca++ can regulate ~[ro by IL-l or TNFa to produce significant amounts of IL-S [10]. keratinocyte terminal differentiation is unknown, increases in cyto­ d thOugh local production of the cytokine at inflamed sites is un- solic free Ca++ may activate a series of regulatory enzyme systems OUbtedly responsible, at least in part, for the cutaneous trafficking that could induce keratinocytes to differentiate irreversibly. Over- 298 TUSCHIL ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY all , the findings reported here suggest that, in the microenviron­ expressed in high levels in psoriatic skin and stimulates proli~eratiox ment of an inflammatory site like a psoriatic lesion, locally produced of cultured human keratinocytes. 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