Murine Epidermal Cell Line Research Articles

Mitigation of Nitrogen Mustard-Induced Skin Injury by the β-Blocker Carvedilol and Its Enantiomers.

The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard (NM) cause severe vesicating skin injuries. The pathologic mechanisms for the skin injury following mustard exposure are poorly understood; therefore, no effective countermeasure is available. Previous reports demonstrated the protective activity of carvedilol, a US Food and Drug Administration (FDA)-approved β-blocker, against UV radiation-induced skin damage. Thus, the current study evaluated the effects of carvedilol on NM-induced skin injuries in vitro and in vivo. In the murine epidermal cell line JB6 Cl 41-5a, β-blockers with different receptor subtype selectivity were examined. Carvedilol and both of its enantiomers, R- and S-carvedilol, were the only tested ligands statistically reducing NM-induced cytotoxicity. Carvedilol also reduced NM-induced apoptosis and p53 expression. In SKH-1 mice, NM increased epidermal thickness, damaged skin architecture, and induced nuclear factor κB (NF-κB)-related proinflammatory genes as assessed by RT2 Profiler PCR (polymerase chain reaction) Arrays. To model chemical warfare scenario, 30 minutes after exposure to NM, 10 μM carvedilol was applied topically. Twenty-four hours after NM exposure, carvedilol attenuated NM-induced epidermal thickening, Ki-67 expression, a marker of cellular proliferation, and multiple proinflammatory genes. Supporting the in vitro data, the non-β-blocking R-enantiomer of carvedilol had similar effects as racemic carvedilol, and there was no difference between carvedilol and R-carvedilol in the PCR array data, suggesting that the skin protective effects are independent of the β-adrenergic receptors. These data suggest that the β-blocker carvedilol and its enantiomers can be repurposed as countermeasures against mustard-induced skin injuries. SIGNIFICANCE STATEMENT: The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard cause severe vesicating skin injuries for which no effective countermeasure is available. This study evaluated the effects of US Food and Drug Administration (FDA)-approved β-blocker carvedilol on nitrogen mustard-induced skin injuries to repurpose this cardiovascular drug as a medical countermeasure.

The β‐blockers carvedilol and nebivolol inhibit neoplastic transformation through a β2‐adrenergic receptor independent mechanism

β‐adrenergic receptors (β‐ARs) are crucial mediators of physiological responses to catecholamines (i.e. epinephrine and norepinephrine), and β‐blockers competitively inhibiting β‐ARs thereby preventing the associated cardiovascular effects of β‐ARs. Aside from their cardiovascular function, β‐blockers are potential chemopreventive agents. Previous studies identified a subset of β‐blockers as potential cancer preventive agents. Carvedilol and nebivolol inhibit EGF‐induced neoplastic transformation of JB6 P+ cells, a non‐cancerous but transformation sensitive murine epidermal cell line. However, the antineoplastic mechanism(s) of action have yet to be elucidated. Therefore, we sought to evaluate the role of the β2‐AR, the only β‐AR present in JB6 P+ cells, in the β‐blocker antineoplastic effect. First, an EGF‐mediated soft agar transformation assay was utilized to evaluate the combined effect of carvedilol with either CGP 12177 or nadolol; CGP 12177 and nadolol lack a cancer preventive effect. 10 μM CGP 12177 (KD= −9.39) had no effect on carvedilol (KD= −9.40) mediated inhibition of EGF‐induced transformation of JB6 P+ cells: −6.48±0.09 IC50 versus −6.67±0.05 IC50 for carvedilol and carvedilol plus CGP 12177, respectively. Similarly, carvedilol combined with 10 μM nadolol (KD= −8.60) resulted in no shift in the concentration‐response curve (−6.55±0.03 IC50 versus −6.50±0.06 IC50). Since carvedilol is a non‐selective β‐AR and α1‐AR antagonist, we assessed the involvement of the α1‐AR by combining the β‐blocker nebivolol, that lacks α1‐AR activity, with 10 μM CGP 12177. Combination of nebivolol (KD= −7.91 for the β2‐AR) and CGP 12177 did not alter the potency of nebivolol (−6.46±0.11 and −6.81±0.12 for nebivolol and nebivolol plus CGP 12177, respectively). To further the pharmacological studies, the efficacy and potency of carvedilol in preventing EGF‐induced transformation of JB6 P+ cells was compared in scramble control shRNA and Adrb2 (β2‐AR) shRNA infected cells. With 72% β2‐AR knockdown, carvedilol prevented EGF‐mediated transformation of JB6 P+ cells (IC50= −6.02±0.07), although with a slightly reduced potency compared to the scramble control (IC50= −6.24±0.06). The data demonstrate that the β‐blocker carvedilol and likely nebivolol function as a cancer preventive agent independently of their widely‐understood action as β‐AR blockers.

Abstract 5252: Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents

Abstract Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong and Paeoniae, is one of the most popular Chinese medicines for women's diseases. Previously we showed that SWT was able to upregulate genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, suggesting a potential application for cancer chemoprevention. The present study examined the chemopreventive activity of SWT using models of skin carcinogenesis. In JB6 P+ cells, a non-cancerous murine epidermal cell line for studying skin tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. In a 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine skin tumorigenesis model, both topical and oral treatment of SWT inhibited epidermal hyperplasia, proliferating cell nuclear antigen expression, and H-ras mutations induced by DMBA treatment. In addition, SWT exhibited a significant antimutagenic activity against DMBA-induced mutagenicity, determined by the Ames Test using Salmonella typhimurium TA100 in the presence of metabolic activator S9 system. To identify active components in SWT, among nine compounds previously reported in commercial SWT products, in silico molecular docking analysis predicted some as potential Nrf2 activators due to an ability of interfering the forming of Nrf2-Keap1 complex. Three of these compounds, gallic acid, Z-liguistilide and senkyunolide A, were confirmed with highest potency of increasing the antioxidant response element luciferase reporter activity, inducing Nrf2-regulated genes Hmox1, Slc7A11 and Nqo1, and inhibiting EGF-induced JB6 P+ transformation. Further mechanistic studies showed that SWT and the three compounds suppressed EGF-induced activation of the activator protein 1 (AP-1), an essential transcription factor involved in skin carcinogenesis. The antimutagenic activity for the three compounds was also confirmed with the Ames Test. In conclusion, these results provide evidence that SWT and its constituents are able to prevent skin cancer, at least partly, by activating the Nrf2 pathway and blocking the activation of AP-1. Thus, this widely used Chinese medicinal formula may provide a promising option toward preventing skin cancer or may be other types of cancer. Citation Format: Kevin Huang, Mandy Liu, Suhui Zhang, Steven Yeung, Andy Chang, Li Qian, Payal Chatterjee, Rui Li, Su Zhou, Nan Mei, Zhijun Wang, Cyrus Parsa, Robert Orlando, Yun Luo, Ying Huang. Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5252.

Differentiation of skin sensitizers from irritant chemicals by interleukin-1α and macrophage inflammatory protein-2 in murine keratinocytes

The development of novel alternative testing methods is required to identify the sensitizing capacity of chemicals as a replacement for animal experimentation. We aimed to evaluate in vitro assays as screening tools for detecting skin sensitizers. The murine epidermal keratinocyte cell line HEL-30 was exposed to 16 relevant skin sensitizers and 6 skin irritants. The dose causing 75% cell viability (CV75) measured by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was chosen as a highest dose and three more doses (0.5×, 0.1×, and 0.01× of CV75) were tested. As parameters, levels of interleukin 1α (IL-1α), macrophage inflammatory protein 2 (MIP-2), IL-6, and IL-18 production were measured using 4 different doses. The accuracy of detecting sensitizers or irritants by IL-1α or MIP-2 alone was exactly same: 75% (12 out of 16) for sensitizers, 83% (5 out of 6) for irritants, and overall 77% (17 out of 22). However, combination of IL-1α and MIP-2 showed better accuracy: 94% (15 out of 16), 67% (4 out of 6), and overall 86% (19 out of 22). IL-6 and IL-18 could not differentiate sensitizers from irritants. This study suggests that the combination of pro-inflammatory cytokines IL-1α and MIP-2 in murine HEL-30 cells can be a reliable in vitro method for identifying chemicals that may act as skin sensitizers.

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

BackgroundSkin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.MethodsWe have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.ResultsCOCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.ConclusionsWe propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Laminin-121—Recombinant expression and interactions with integrins

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, β2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its Tm value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the β chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6β1 and α7β1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the β2 laminins have higher affinity for integrins than the β1 laminins.

Assessment of potency of allergenic activity of low molecular weight compounds based on IL-1α and IL-18 production by a murine and human keratinocyte cell line

Assessment of allergenic potency of low molecular weight compounds is generally performed using animal models, such as the guinea pig maximisation test and the murine local lymph node assay (LLNA). Progress in unravelling the mechanisms of skin sensitisation, including effects on the production of cytokines by the different cell types of the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitisation testing. The aim of the present study was to establish an in vitro method to assess the potency of allergens, on the basis of their induction of cytokine production by murine and human keratinocytes. In the present study we used test systems comprised of the murine epidermal keratinocyte cell line HEL-30 and the human keratinocyte cell line HaCaT. We exposed these cell lines to the allergens ethyl- p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), and phthalic anhydride (PA). IL-1α and IL-18 dose–response data were evaluated by non-linear regression analysis and at a stimulation index of 3 of cytokine production of treatment versus control, the corresponding allergen concentration was calculated. For HEL-30, for both cytokines DNCB showed the strongest potency followed in this order by PA, benzocaine, and DEA. This classification was similar to our previous findings obtained in the LLNA. For HaCaT, unfortunately, such ranking proved to be much less feasible. In conclusion, to assess the potency of allergens the murine keratinocyte cell line HEL-30 may be a useful in vitro test system, alternative to in vivo models, although this requires further testing using a much wider range of compounds.

UVB radiation dysregulates T cell-mediated secretion of IL-1β and IL-6 by a murine epidermal dendritic cell line (XS52)

UVB radiation dysregulates T cell-mediated secretion of IL-1β and IL-6 by a murine epidermal dendritic cell line (XS52)

Cadherin/catenin complexes in murine epidermal keratinocytes: E-cadherin complexes containing either beta-catenin or plakoglobin contribute to stable cell-cell contacts.

The cadherin/catenin complexes expressed by a murine epidermal keratinocyte cell line PDV, expressing E- and P-cadherin, have been analysed using a combination of biochemical and confocal microscopy analysis. Two types of E-cadherin complexes, containing beta-catenin or plakoglobin and alpha-catenin, were detected in PDV cells as in other cell types, while P-cadherin was mainly detected in complexes containing beta-catenin and alpha-catenin in PDV and other murine epidermal keratinocytes. Biotin-labelling studies have shown that both types of E-cadherin complexes are present at the surface of confluent cells. Furthermore, confocal microscopy analysis indicated that E-cadherin/plakoglobin complexes are located in stable cell-cell contacts at the middle lateral membranes and associated with alpha-catenin and the actin cytoskeleton, with a similar distribution to that to the E-cadherin/beta-catenin complexes. In addition, E-cadherin/plakoglobin complexes not associated with alpha-catenin or the actin cytoskeleton were detected in lower planes of the lateral contacting membranes as well as E-cadherin non-associated with catenins in the more basal planes. These studies support that in murine epidermal keratinocytes both beta-catenin- and plakoglobin-containing E-cadherin complexes contribute to the maintenance of stable cell-cell contacts and suggest a differential role of the plakoglobin containing complexes in different epithelial cell types.

Requirement for phosphatidylinositol 3-kinase in epidermal growth factor-induced AP-1 transactivation and transformation in JB6 P+ cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in a variety of biological processes, including regulation of gene expression, cell growth, and differentiation. However, little is known about its role in the cytoplasmic events involved in epidermal growth factor (EGF)-induced transduction of signals to the transcriptional machinery of the nucleus and in EGF-induced cell transformation. In this study, we examined whether PI 3-kinase is a mediator for the activation of AP-1 and neoplastic transformation by EGF in the murine epidermal cell line JB6. The results showed the following. (i) EGF not only induced a high level of PI 3-kinase activity by itself but also enhanced insulin-induced PI 3-kinase activity in JB6 P+ cells, the EGF-induced PI-3 kinase activity could be blocked by constitutive overexpression of a dominant negative P85 subunit of PI 3-kinase (deltaP85), and insulin could markedly promote EGF-induced AP-1 activity in a dose-dependent manner in JB6 P+ cells as well as promote EGF-induced JB6 P+ cell transformation. (ii) Inhibition of PI-3 kinase with wortmannin or LY294002 markedly decreased the AP-1 activity induced by insulin, EGF, or EGF and insulin in a dose-dependent manner, while wortmannin did not block UVB-induced AP-1 activity. (iii) AP-1 activation by insulin, EGF, or EGF and insulin could be completely inhibited by overexpression of deltaP85 in all the dose and time courses studied. (iv) Inhibitors of PI 3-kinase (wortmannin and LY294002) and stable overexpression of deltaP85 inhibited EGF-induced transformation but had no significant inhibitory effect on cell proliferation induced by EGF or EGF and insulin. These results demonstrate for the first time that PI 3-kinase appears to be required for EGF- or insulin-induced AP-1 transactivation and cell transformation but not cell proliferation in JB6 cells.

Ultraviolet B radiation sensitizes a murine epidermal dendritic cell line (XS52) to undergo apoptosis upon antigen presentation to T cells.

Ultraviolet B irradiation of skin leads to immunologic tolerance, rather than immunity against newly introduced Ag, by altering the function of Langerhans cells, skin-specific members of the dendritic cell (DC) family. Using the murine epidermal-derived DC line, XS52, which retains important features of resident Langerhans cells, we have tested the hypothesis that UV radiation delivers a signal leading to apoptosis. XS52 cells, when exposed to modest fluences (25-100 J/m2) of radiation, underwent apoptosis during a subsequent 6-h incubation with LPS or upon 6-h coculture with the keyhole limpet hemocyanin-specific Th1 clone HDK-1 in the presence of Ag. Specifically, XS52 cells treated in this way exhibited diminished cell viability, DNA laddering, and condensed staining of DNA. By contrast, none of these changes was induced by radiation alone, LPS alone, or coculture with T cells and Ag. Likewise, neither UV radiation plus T cells nor radiation plus Ag were sufficient to induce apoptosis, indicating that both T cells and Ag are required to induce apoptosis in the UV-sensitized cells. XS52 cells remained fully susceptible to T cell-mediated apoptosis even 16 h after irradiation, indicating the persistence of the sensitized state. These observations establish a model in which UV radiation induces a first event in which DC become sensitive to a second, apoptotic signal that is delivered by Ag-specific interaction with T cells or by LPS. We suggest that DC undergoing apoptosis deliver unusual activation signals to T cells during Ag presentation, signals that lead to cellular unresponsiveness rather than to effective immunity.

Effect of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol, on protein content, protein synthesis and LDH leakage in mouse epidermal cells

The cytotoxicity of 25-hydroxycholesterol, 26-hydroxycholesterol and its analogue, 26-aminocholesterol was investigated in murine epidermal cell line, HEL-30. Lactate dehydrogenase (LDH) leakage, protein synthesis and protein content were determined after exposure of cell monolayers to the compounds ranging from 0.1–200 μM for 2, 6 or 24 h. 26-Aminocholesterol affected all the parameters studied time and concentration dependently; 25-hydroxycholesterol and 26-hydroxycholesterol were not toxic for HEL-30 cells. The cellular target for 26-aminocholesterol was primarily the membrane, since the LDH leakage was already detectable 10 min after exposure. On the other hand, for the other two oxysterols a protective role on this structure might be postulated. In fact 25-hydroxycholesterol and 26-hydroxycholesterol decreased the natural LDH leakage due to the ageing of the culture. In addition, 20 μM 25-hydroxycholesterol reversed the effect of moderately lytic doses of 26-aminocholesterol and Triton X-100, but not of sodium dodecyl sulfate.

The predominant role of surfactants in the modulation of toxicity of detergent products: An in vitro analysis of shampoos

20 shampoos and their component surfactants were studied in vitro using a murine epidermal cell line (HEL/30). Lactate dehydrogenase (LDH) leakage into the cell medium and the inhibition of protein neosynthesis were evaluated as markers of cytotoxicity after 2 hr of exposure. The coefficients of correlation were −0.83 and −0.69 for comparisons between total surfactant content and inhibition of protein synthesis and leakage of LDH, respectively. There was an inverse relationship between the inhibition of protein synthesis and the amphoteric surfactant content of the shampoo. An excellent correlation ( r = 0.94) was found between LDH leakage values and scores from eye irritation tests in vivo on seven of the shampoos. The results obtained suggest that this model allows quantitative evaluation of the cytotoxicity of detergent products (shampoos) and of the contribution of their major components (surfactants) to that toxicity.

Cell cultures: A tool for the study of mechanisms of toxicity

Many toxic agents can affect cellular function by selectively interacting with cytoskeletal components, possible cell targets of toxicity. This phenomenon may represent the initial event in altering cell metabolism. The toxic effect of tributyltin chloride (TBT) on ATP levels, protein synthesis, glutathione (GSH) levels and lactate dehydrogenase (LDH) activity was investigated in a murine epidermal cell line (HEL-30). Recovery of cell functions was observed either when metabolism was induced by the addition to the incubation medium of an S-9 preparation, or when dithiothreitol (DTT) was added to the medium 5 min after the damage and in the absence of TBT. The level of F-actin, a cytoskeletal component, was lowered in resting human neutrophils by TBT and triphenyltin chloride (TPT) but not by SnCl 2; moreover, the cellular response to a polymerizing stimulus such as fMLP, a chemotactic peptide, was no longer detectable after TBT or TPT treatment. The evidence that neutrophils of a subject who had been acutely exposed to TPT did not show the normal actin polymerization response to chemotactic stimulus (Colosio et al., British Journal of Industrial Medicine 1991, 48, 136) suggests a possible use of this function as a marker of human exposure to defined toxic chemicals.

ATP and Protein Synthesis Assays to Evaluate the Toxicities of Preservatives in Vitro

Cellular protein content, protein synthesis, ATP level and lactate dehydrogenase (LDH) activity, measured in a murine epidermal cell line (HEL/30), were used as the endpoints for determining the cytotoxicities of 17 antimicrobial chemicals. The relative toxicities of the test compounds were quantified by the determination of the concentrations inducing a 50% inhibition of [3H]-leucine incorporation into proteins (IC50), causing a 50% reduction of ATP level or final cell protein content or producing the maximal effect on LDH leakage (EC50) after 2 hours of treatment. The results indicate a good correlation between both the reduction of ATP level and inhibition of protein synthesis and the minimal inhibitory concentration (MIC) on different microorganisms, suggesting that ATP and protein synthesis assays could be useful as prescreening methods for testing the cytotoxicities of preservatives.

Reversibility of tributyltin-chloride-induced protein synthesis inhibition after ATP recovery in HEL-30 cells

The effect of tributyltin chloride (TBT) on ATP levels and protein synthesis was investigated in a murine epidermal cell line (HEL-30). Five minutes after exposure to 10 −5 M TBT, cellular levels of ATP decreased by 57% and protein synthesis was significantly inhibited. The effect on ATP was stable up to 2 h incubation, whereas inhibition of protein synthesis increased with time of treatment. Partial (1 h) or complete (2 h) recovery of the two cell functions was observed when dithiothreitol (DTT) was added to the medium, 5 min after the damage and in the absence of TBT. The sequence of the observed effects suggests that TBT inhibition of protein synthesis is due to impairment of ATP production.

Quantification ofIn VitroCytotoxicity of Surfactants: Correlation with their Eye Irritation Potential

A number of surfactants, including representatives of the cationic, anionic, nonionic, and amphoteric groups, were studied for their effects on lactate dehydrogenase (LDH), protein content, and de novo synthesis, at concentrations ranging from 1 to 500 µg/d medium, in a murine epidermal cell line (HEL/30). Protein synthesis, evaluated as [3H]leucine incorporation into cell proteins, was a more sensitive endpoint of toxicity than LDH leakage or protein content. Comparative surfactant toxicity followed the general order of cationic > anionic > nonionic > amphoteric. A good rank correlation was observed between their relative toxicity produced in vitro and the eye irritation produced in vivo in the Drake test. This sensitive and reproducible in vitro method may offer a means for screening potentially imtating water-soluble chemicals such as surfactants to quantify their toxicity.

Arachidonic acid metabolism in HEL/30 murine epidermal cell line.

The established mouse epidermis-derived cell line HEL/30 was incubated in the presence of 3H arachidonic acid (AA) for 1 h. After medium removal, cells were reincubated with fresh medium in the presence or absence of the calcium ionophore A23187 and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The AA metabolites formed were extracted from cell-free medium and analyzed using TLC and HPLC. The distribution of the recovered radioactivity showed PGE2, 15-hydroxy-eicosatetraenoic acid (15-HETE), and leukotriene B4 (LTB4), as major products of AA metabolism. The presence of calcium ionophore A23187 increased the release of radioactivity, without affecting the profile of metabolites present in the medium. TPA elicited a preferential increase of cycloxygenase metabolism, this effect being reversed by indomethacin. 5,8,11,14-eicosatetraynoic acid (ETYA) almost completely inhibited LT and HETE formation in A23187 and TPA-treated cells. The results show that HEL/30 cells are able to metabolize AA via both cyclo- and lipoxygenase pathways and that these activities can be modified by chemical means. This cell line might be a suitable tool for studying the involvement of arachidonic acid cascade in cell response to exogenous stimuli.

Lymphokine mediated production of an epidermal cell proliferation factor by cultured murine bone marrow cells.

The effect of cultured bone marrow cell supernatant (BMS) was studied on the proliferative response of cells of the transformed murine epidermal cell line Pam 212. Elevated DNA synthesis was found in Pam 212 cells cultured with BMS from bone marrow cells grown in spleen cell conditioned medium with concanavalin A (ConA). Pam cell proliferative activity was related to the histamine content in the culture supernatant. Neither spleen cell conditioned medium with IL1, IL2, or IL3 activity, nor ConA alone, showed any effect on Pam cell growth. A soluble mediator from the cultured bone marrow cells with mast cell characteristics was thought to be responsible for the stimulation of Pam cell growth, and Con A appeared to be a prerequisite for generation of this factor by the bone marrow cells.

High-performance liquid chromatographic separation of distinct epidermal cell-derived cytokines

High-performance liquid chromatography (HPLC) is useful for the purification and separation of immunoregulatory cytokines, such as macrophage-derived interleukin 1 (IL 1). In addition to macrophages, epidermal cells also release a mediator, epidermal cell (EC) derived thymocyte-activating factor (ETAF), whcih cannot be separated from IL 1. Moreover, it has been shown recently that EC produce a distinct interleukin 3-like mast cell-activating factor (EC IL 3). This study was performed to investigate whether HPLC may be useful for the separation of EC-derived cytokines, such as ETAF and EC IL 3. For factor production, a murine EC line (Pam 212) was used. ETAF activity was measured using the thymocyte costimulator assay. EC IL 3 was was determined by induction of the proliferative activity of an IL 3-dependent cell line (32 DCL). Using a TSK 125 size-exclusion column and phosphate-buffered saline (pH 7.2) as the mobile phase, ETAF was eluted with an apparent molecular weight of 17 kD, and EC IL 3 with a molecular weight of 28 kD. When EC supernatants were chromatofocused on a Mono P column, ETAF activity was eluted with apparent p I values of 6.8, 6.2 and 5.3, and ECL IL 3 activity with p I 7.8, 7.4 and 7.1. When reversed-phase HPLC (RP-HPLC) (equilibration with water and a 0–100% concave acetonitrile gradient) was applied ETAF exhibited four distinct peaks, whereas EC IL 3 was eluted as one major peak between 70 and 80% acetonitrile. Separation on a Bio-Gel HPHT column with a sodium phosphate gradient was not satisfactory, but the recovery was high. It is concluded that chromatofocusing on Mono P and RP-HPLC are suitable methods for the separation of cytokines, such as ETAF and EC IL 3, both of which are produced by EC.