Effects of tumour necrosis factor alpha and interleukin 1 beta on the proliferation of cultured glomerular epithelial cells

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor alpha (TNF alpha) or recombinant interleukin 1 beta (IL-1 beta) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1-50 ng/ml of TNF alpha, growth being up to 400% more than in the control culture. The effect of TNF alpha depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF alpha, IL-1 beta inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF alpha and IL-1 beta and between TNF alpha and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.     

Production of IL-1 alpha and IL-1 beta by human skin equivalents parasitized by Sarcoptes scabiei

Human skin equivalents (HSEs) were used as a model to investigate interleukin (IL)-1 alpha and IL-1 beta secretions by keratinocytes stimulated by Sarcoptes scabiei (SS). SS mites burrowed into the stratum corneum when placed on the surface of cultured HSEs. Mites lived for 14 days. Mites and mite products induced cells in the HSEs to secrete IL-1 alpha and IL-1 beta within 16 hr. Scabies mites induced production of greater amounts of IL-1 alpha than IL-1 beta. Hepatocyte growth factor in the culture medium at 3 and 30 ng/ml upregulated the secretions of both IL-1 alpha and IL-1 beta by mite-infested skin equivalents, whereas 10 ng/ml of IL-6 upregulated production of only IL-1 beta. Therefore, these cytokines were important immunomodulating factors influencing keratinocyte secretion of IL-1 alpha and IL-1 beta in vitro. The results of this study provide the first evidence that keratinocytes (possibly fibroblasts) in the skin produce these cytokines in response to scabies mites or other ectoparasitic arthropods. Because IL-1 alpha and IL-1 beta are potent inducers of inflammation and keratinocytes are among the first effector cells to encounter scabies mites and their products, these cells may be key initiators of the inflammatory/immune reaction to scabies.     

Angiotensin II stimulates proliferation of primary human keratinocytes via a non-AT1, non-AT2 angiotensin receptor

Angiotensin II is a hormone with long known cardiovascular actions. Recent studies revealed an additional role for angiotensin II in the regulation of cell proliferation. This study was performed to clarify whether skin is a target organ for these novel angiotensin actions. Radioligand binding studies identified a high-affinity angiotensin receptor on human primary keratinocytes in vitro with a Kd of 4.5 nM and a Bmax of 0.12 nM. Competition experiments with losartan and PD 123177 revealed that this receptor was not of the AT1- nor the AT2-subtype. Stimulation of human keratinocytes with angiotensin II (10(-10) to 10(-5) M) led to a dose dependent increase in 3H-thymidine incorporation, indicating that the keratinocyte receptor mediates a mitogenic effect. This effect is comparable at 10(-9) M to stimulation of keratinocytes by EGF (50ng/ml) and FGF (50ng/ml). These results demonstrate for the first time a possible physiological role for angiotensin II in human skin involving the regulation of keratinocyte proliferation.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.     

Cell type-specific modulation of cell growth by transforming growth factor beta 1 does not correlate with mitogen-activated protein kinase activation

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional cytokine that positively or negatively regulates the proliferation of various types of cells. In this study we have examined whether or not the activation of the mitogen-activated protein (MAP) kinases is involved in the transduction of cell growth modulation signals of TGF-beta 1, as MAP kinase activity is known to be closely associated with cell cycle progression. Although TGF-beta 1 stimulated the growth of quiescent Balb 3T3 and Swiss 3T3 cells, it failed to detectably stimulate the tyrosine phosphorylation and activation of the 41- and 43-kDa MAP kinases at any time point up to the reinitiation of DNA replication. TGF-beta 1 also failed to stimulate the expression of the c-fos gene. Furthermore, TGF-beta 1 synergistically enhanced the mitogenic action of epidermal growth factor (EGF) without affecting EGF-induced MAP kinase activation in these fibroblasts, and it inhibited the EGF-stimulated proliferation of mouse keratinocytes (PAM212) without inhibiting EGF-induced MAP kinase activation. Thus, the ability of TGF-beta 1 to modulate cell proliferation is apparently not associated with the activation of MAP kinases. In this respect, TGF-beta 1 is clearly distinct from the majority, if not all, of peptide growth factors, such as platelet-derived growth factor and EGF, whose ability to modulate cell proliferation is closely associated with the activation of MAP kinases. These results also suggest that the activation of MAP kinases is not an absolute requirement for growth factor-stimulated mitogenesis.     

A granisetron-droperidol combination prevents postoperative vomiting in children

M proteins are receptor proteins and one of the virulence factors of streptococci. M proteins seem to play a role in inflammatory skin disorders such as psoriasis. It is however unknown whether M proteins have a direct influence on proliferative activity of human keratinocytes. In the present study human HaCaT keratinocytes were exposed to M proteins (M1, M3, M5, M12) and the proliferative and proinflammatory response was analyzed. We found a dose-dependent inhibition of keratinocyte proliferation with crude extract of strain M3 4/55. Following affinity chromatography we found inhibitory activity for keratinocyte proliferation with a maximum of 80% at 10-8 M in the M protein. Additionally tested M1 protein preparation showed an inhibitory activity of 55% whereas other M preparations (5 and 12) did not show any effect. In supernatants from HaCaT cultures IL-1alpha, IL-1beta, IL-6, IL-8, TNFalpha and ICAM-1 were measured by ELISA. The levels of IL-8 were high and TNFalpha was upregulated, whereas ICAM-1 was decreased from around 20 ng/ml to almost zero. In contrast to the streptococcal-derived M3 protein preparation the recombinant M3 did not interfere with the proliferation of HaCaT cells. Because neither recombinant M3 protein nor M3 protein purified by ion exchange chromatography on a Q-resource column had any antiproliferative activity on keratinocytes we suggest, that a component different from M3 protein was responsible.     

A possible mechanism for initiation of lipid peroxidation by ascorbate in rat liver microsomes

RRR-alpha-tocopheryl succinate (VES) was studied for effects on murine EL-4 cell proliferation and production of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta). VES was biphasic in its actions: 0.1 microgram/ml enhanced EL-4 cell proliferation, whereas 10-20 microgram/ml inhibited cellular proliferation. Cell-conditioned media (CM) from EL-4 cells treated with 0.2 ng/ml phorbol myristate acetate (PMA) + 0.1 microgram/ml VES contained increased amounts of IL-2, as determined by the murine cytotoxic T cell IL-2-dependent CTLL-2 bioassay. VES at 0.1 microgram/ml or 0.1 microgram/ml VES + 0.2 ng/ml PMA induced the expression of IL-2 mRNA by EL-4 cells three to nine hours after treatment. CM from EL-4 cells treated with VES at 10-20 microgram/ml exhibited potent antiproliferative activity when tested in the TGF-beta-responsive mink lung cell (Mv1Lu) bioassay and showed reduced inhibitory effects when tested on TGF-beta receptor-negative mink lung (DRA-27) cells. CM from control-treated EL-4 cells exhibited no antiproliferative activity. The VES-induced antiproliferative activity was characterized as TGF-beta by neutralization analyses and immunoprecipitation of metabolically labeled proteins with TGF-beta-specific reagents. VES treatment of EL-4 cells had no effect on TGF-beta 1 mRNA expression while downregulating TGF-beta 3 mRNA expression. In summary, these studies showed that 0.1 microgram/ml VES enhanced cellular proliferation, in part, via increased IL-2 production, whereas 10-20 micrograms/ml VES inhibited cellular proliferation, in part, via the secretion of biologically active TGF-beta.     

The effect of epidermal growth factor on the growth potential of epithelial cells from human lens

The growth promoting effect of epidermal growth factor (EGF) was studied in cultures of epithelial cells from human normal and cataractous lenses. The growth potential of lens epithelial cells was measured by MTT assay. The concentration of EGF in culture medium were classified into 7 groups (0 ng/ml-10(3) ng/ml). When the concentration of EGF was 1 ng/ml, EGF induced the highest increase of growth potential epithelial cells compared with an EGF-free group.     

The reversed-flow medio-distal fasciocutaneous island thigh flap: anatomic basis and clinical applications

To investigate changes in retinal pigment epithelial (RPE) cells during wound healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every 12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor- beta2 (TGF-beta2) to cultures at concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells were distributed in concentric rings that moved centripetally over time and corresponded to the leading edge of the area stained with anti-ECM antibodies. TGF-beta2 enhanced ECM deposition, but EGF and PDGF did not. TGF-beta2 decreased the healing rate in a dose-dependent manner, whereas PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM components may be produced in a specific sequence during healing. TGF-beta2 may promote RPE cell differentiation, and PDGF may enhance proliferation during wound healing of the RPE.     

Calcipotriol (MC 903), a synthetic derivative of vitamin D3 stimulates differentiation of squamous carcinoma cell line in the raft culture

We investigated whether calcipotriol, a synthetic derivative of vitamin D3 has the ability to correct defects in the control of proliferation and differentiation of human squamous carcinoma cells using the raft culture of SCC 13 cell line. Calcipotriol treatment at concentrations of 10(-8)-10(-6) M considerably enhanced terminal differentiation of SCC 13 cells, as shown by the appearance of enucleated-eosinophilic cells as well as granular cells in their upper cell layers. Immunohistochemical staining showed marked increases in the differentiation of marker proteins such as keratin 1, involucrin, or filaggrin expressing cells in their upper layers. The elevated expression at protein level was confirmed by immunoblotting analysis. Furthermore, calcipotriol also stimulated basal cell marker proteins such as keratin 14 and EGF receptor. However, the numbers of basal marker expressing cells within the architecture of SCC 13 raft culture were markedly reduced upon calcipotriol treatment, and their localization was mainly restricted in the innermost cell layer. In addition, calcipotriol stimulated EGF receptor biosynthesis for the first 16 hours post treatment and subsequently inhibited [3H]-thymidine incorporation of SCC 13 cells at 24 hours. In this study, we have clearly demonstrated that the long term application of calcipotriol considerably improves the complex defects in the regulation of proliferation and differentiation of SCC 13 cells, as supported by morphological and biochemical observations. This provides an evidence that calcipotriol can be applied clinically as a potent differentiation inducer in the treatment of human squamous cell carcinoma.     

Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells

The aim of this study was to elucidate some of the possible mechanisms of action of the vitamin D analogue calcipotriol in vivo. Calcipotriol is finding increasing use in the treatment of psoriasis, but the primary target cell in vivo has not yet been identified. We treated psoriatic patients and healthy volunteers with calcipotriol and placebo ointment for 4 and 7 days, and obtained epidermal cell suspensions from treated areas. Epidermal cells were cocultured with autologous T cells, isolated from peripheral blood, in the absence or the presence of a classical antigen or a superantigen. In both psoriatic and normal skin, calcipotriol treatment did not alter the capacity of epidermal antigen-presenting cells to stimulate the proliferation of autologous T cells, either in the absence or in the presence of exogenous antigen. Epidermal cell suspensions were analysed further by staining for infiltrating leucocytes (CD45+) and Langerhans cells (CD1a+). Flow cytometric analysis showed that calcipotriol did not alter the number of CD45+ cells or Langerhans cells in psoriatic skin. These results indicate that calcipotriol does not alter either the number of the function of epidermal antigen-presenting cells in psoriatic epidermis. In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry. More specifically, we stained for CD29+ keratinocytes and found an even more significant reduction in proliferative capacity. This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis. In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis, rather than via an effect on infiltrating leucocytes, including antigen-presenting cells.     

Effects of interferons on tumour necrosis factor alpha production from human keratinocytes

Interferons (IFNs) have been reported to have pleiotrophic effects including the ability to induce the production of other cytokines in several cell types. Tumour necrosis factor alpha (TNF-alpha) is pro-inflammatory cytokine a known to be produced by a variety of cells including human keratinocytes. In the present study, we sought to determine the effects of IFNs on TNF-alpha production from human keratinocytes. IFN-gamma (50-100 ng/ml) induced TNF-alpha production dose dependently, but no induction of TNF-alpha was observed with IFN-alpha or IFN-beta. Since in the epidermis cytokines often work with in a cascade fashion and keratinocytes are a source of primary cytokine, IL-1 alpha, whether combined treatment with IFN-gamma and IL-1 alpha had a synergistic effect on TNF-alpha production was examined. Combined treatment with IFN-gamma (100 ng/ml) and IL-1 alpha (10 ng/ml) induced 2-3-fold higher level of TNF-alpha than IL-1 alpha alone. These results suggest that IFN-gamma is a positive regulator for the production of TNF-alpha from human keratinocytes and likely to increase skin inflammation.     

Modulation of transforming growth factor-beta 1 effects by cytokines

The purpose of the present study was to determine the effects of human recombinant transforming growth factor-beta 1 (TGF-beta 1) on the proliferation of normal cell and cancer cell lines and to evaluate the mechanism of TGF-beta-induced immunosuppression. Murine H238 fibrosarcoma and human UC-11 glioblastoma cells showed no proliferative change in the presence of TGF-beta, whereas the growth of human LS174T colon adenocarcinoma cells was significantly enhanced at the lower concentrations of TGF-beta. In contrast, Mono/Mac-6, a human monocyte cell line, human peripheral blood mononuclear (PBMN) cells, and BALB/c mouse spleen cells were significantly suppressed by 2.5 to 250 ng/ml of TGF-beta. In order to investigate the mode of action, TGF-beta and other cytokines were added 0, 1, and 2 days after initiation of the culture. Mono/Mac-6 cells showed that 2 days are needed for TGF-beta-induced suppression. Simultaneous addition of TGF-beta and tumor necrosis-alpha (TNF-alpha; 600 units/ml) to Mono/Mac-6 cells resulted in nearly complete suppression by day 3. IL-2, and to a lesser extent IL-4, was able to counteract the suppressive effects of TGF-beta on mitogen-stimulated spleen cells. However, our results indicate that IL-2 is not as effective in restoring responsiveness once T cell activation is well underway. IL-1 and interferon-gamma had no effects on TGF-beta-mediated immunosuppression. Since TGF-beta depressed normal cell growth and since IL-2 could effectively counteract the suppression, we assayed for IL-2 production. When normal spleen cells were treated with 2.5 ng of TGF-beta/ml, a 3.4-fold decrease in IL-2 production was observed. This is a potential mechanism for TGF-beta-mediated immunosuppression.     

Regulation of a novel immediate early response gene, IEX-1, in keratinocytes by 1alpha,25-dihydroxyvitamin D3

1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3] regulates cellular growth and differentiation. We show that in keratinocytes, 1alpha, 25(OH)2D3 reduces concentrations of the messenger RNA of IEX-1, the product of which blocks Fas- or tumor necrosis factor type alpha-induced apoptosis in various cells. In sub-confluent keratinocyte cultures, the addition of 1alpha,25(OH)2D3, in amounts that induce growth arrest, reduces IEX-1 mRNA concentrations. In confluent cells, 1alpha,25(OH)2D3 initially reduces and then increases IEX-1 mRNA concentrations. IEX-1 protein is localized in the nucleus and perinuclear region of keratinocytes. In sub-confluent cells, 1alpha,25(OH)2D3 translocates IEX-1 protein from the nucleus to the perinuclear region and cytoplasm. Since IEX-1 has recently been shown to regulate cell survival and number, we suggest that IEX-1 may play a role in keratinocyte growth and differentiation and that 1alpha,25(OH)2D3 may reduce keratinocyte growth via a reduction in IEX-1 mRNA and a change in the intracellular distribution of IEX-1 protein.     

Interhelical contacts are required for the helix bundle fold of apolipophorin III and its ability to interact with lipoproteins

Skin cancer is the most common human malignancy and is strongly associated with exposure to ultraviolet radiation (UVR). Several mechanisms including an increase in immediate early gene activation have been postulated to be involved in UVR-mediated carcinogenesis. We show that in a dose-dependent manner, UVR induces the expression of messenger RNA of a novel immediate early response gene, IEX-1, in human keratinocytes. Human keratinocytes and mouse fibroblasts transfected with an expression plasmid for IEX-1 grow at a faster rate than keratinocytes transfected with a similar plasmid that does not contain the IEX-1 sequence. IEX-1 protein is localized predominantly in the nucleus of keratinocytes by fluorescent antibody methods and by examination of the location of a green fluorescence IEX-1 fusion protein. Epidermal growth factor (EGF), a major mitogen of keratinocytes, and a tumor-promoting phorbol ester increase IEX-1 mRNA expression. IEX-1 may play a role in keratinocyte proliferation especially following UVR.