Effect of obesity on pulmonary inflammation induced by acute ozone exposure: role of interleukin-6

To determine the role of interleukin (IL)-6 in the increased ozone (O3)-induced inflammation and injury observed in obese vs. lean mice, lean wild-type and leptin-deficient obese (ob/ob) mice were injected with anti-IL-6 antibody (Ab) or isotype control Ab 24 h before exposure to either O3 (2 ppm for 3 h) or room air. Four or 24 h after O3 exposure, bronchoalveolar lavage (BAL) was performed, and the lungs were harvested for Western blotting. Anti-IL-6 Ab caused substantial reductions in O3-induced increases in BAL IL-6 in mice of both genotypes. Four hours following O3, ob/ob mice had increased BAL neutrophils compared with controls, and anti-IL-6-Ab virtually abolished this difference. At 24 h, O3-induced increases in BAL protein and BAL serum albumin were augmented in ob/ob vs. wild-type mice, and anti-IL-6 Ab ablated these obesity-related differences in epithelial barrier injury. O3 increased tyrosine phosphorylation of STAT-3 and STAT-1. There was no effect of obesity on STAT-3 phosphorylation, whereas obesity decreased STAT-1 expression, resulting in reduced STAT-1 phosphorylation. IL-6 neutralization did not alter STAT-3 or STAT-1 phosphorylation in ob/ob or wild-type mice. O3 increased BAL leukemia inhibitory factor (LIF) to a greater extent in obese than in lean mice, and LIF may account for effects on STAT phosphorylation. Our results suggest that IL-6 plays a complex role in pulmonary responses to O3, a role that differs between wild-type and ob/ob mice. Moreover, obesity-related differences in activation of STAT proteins may contribute to some of the differences in the response of obese vs. lean mice.     

Macrophage colony-stimulating factor production by murine and human keratinocytes. Enhancement by bacterial lipopolysaccharide

CSF have a broad range of effects on differentiated cells outside the bone marrow. Site-specific elaboration of these factors may influence local immune reactions. Keratinocytes have been demonstrated to produce a number of immunoactive cytokines, including factors capable of modifying macrophage function. We have previously identified at least two products of keratinocytes that induce DNA synthesis by elicited peritoneal macrophages; one factor has been identified as granulocyte-macrophage CSF. In the present study, the second keratinocyte product has been characterized and identified as macrophage-CSF (M-CSF). Conditioned media from cultures of normal human keratinocytes and the transformed murine keratinocyte cell line PAM 212 induce formation of macrophage colonies in soft agar as well as dose-dependent proliferation of the M-CSF-dependent cell line BAC1.2F5. The bioactivity in both assays is blocked by neutralizing anti-M-CSF antibody. Western blot analysis of cell lysates from both PAM 212 and normal human keratinocytes demonstrates multiple molecular mass forms of M-CSF (45 to 98 kDa). Northern blot analysis (PAM 212 cells) and in situ hybridization (normal keratinocytes) demonstrate expression of M-CSF mRNA. Stimulation of keratinocytes with LPS increases M-CSF synthesis as measured both by bioactivity and level of mRNA expression. Thus, both murine and human keratinocytes produce M-CSF in vitro. Furthermore, production of keratinocyte-derived M-CSF is increased by bacterial LPS. CSF production by keratinocytes may play an important role in regulating the cutaneous immune response.     

miR‐221 promotes keratinocyte proliferation and migration by targeting SOCS7 and is regulated by YB‐1

Proliferation and migration of keratinocytes are vital processes for the successful epithelization specifically after wounding. MiR‐221 has been identified to play a potential role in promoting wound regeneration by inducing blood vessel formation. However, little is known about the role of miR‐221 in the keratinocyte proliferation and migration during wound healing. An in vivo mice wound‐healing model was generated; the expression levels of miR‐221 were assessed by qRT‐PCR and fluorescence in situ hybridization. Initially, we found that miR‐221 was upregulated in the proliferative phase of wound healing. Further, in an in vivo wound‐healing mice model, targeted delivery of miR‐221 mimics accelerated wound healing. Contrastingly, inhibition of miR‐221 delayed healing. Additionally, we observed that overexpression of miR‐221 promoted cell proliferation and migration, while inhibition of miR‐221 had the opposite effects. Moreover, we identified SOCS7 as a direct target of miR‐221 in keratinocytes and overexpression of SOCS7 reversed the effects of miR‐221 in HaCaT keratinocytes. Finally, we identified that YB‐1 regulates the expression of miR‐221 in HaCaT keratinocytes. Overall, our experiments suggest that miR‐221 is regulated by YB‐1 in HaCaT keratinocytes and acts on SOCS7, thereby playing an important role in HaCaT keratinocyte proliferation and migration during wound healing.     

The expression and modulation of IL-1 alpha in murine keratinocytes

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.     

Thr308 determines Akt1 nuclear localization in insulin-stimulated keratinocytes

Here, we determined the localization and activation of protein kinase B (Akt) in acute cutaneous wound tissue in mice. Akt1 represented the major Akt isoform that was expressed and activated in wound margin keratinocytes and also in the cultured human keratinocyte line HaCaT. Mutation of Akt1 protein, exchanging the activation-essential Ser473 and Thr308 residues for inactive Ala or phosphorylation-mimicking Asp and Glu residues, revealed that phosphorylation of Ser473 represented an essential prerequisite for auto-phosphorylation of Thr308 within the Akt1 protein in keratinocytes. Moreover, cell culture experiments and transfection studies using Thr308 mutated Akt1 proteins demonstrated that phosphorylation of Akt1 at Thr308 appeared to selectively exclude the active kinase from the nucleus and direct the kinase to the cytoplasmic compartment in keratinocytes upon insulin stimulation. In summary, our data show that phosphorylation of Thr308 during insulin-mediated Akt1 activation is an essential prerequisite to exclude Akt1 from the nuclear compartment.     

HSP25 is involved in two steps of the differentiation of PAM212 keratinocytes

HSP25 is a member of the small heat shock protein family. This 25-kDa protein exhibits a highly specific distribution during mouse embryonic development. Although multiple functions have been proposed for HSP25, the role it plays during differentiation is still unknown. High levels of HSP25 can be detected in embryonic and adult skin. During epidermis differentiation, the concentration of HSP25 increases with the distance of keratinocytes from the basal layer, in parallel with the extent of keratinization. We used an ex vivo cellular system, PAM212 cells, to analyze quantitatively and qualitatively the dynamics of HSP25 production and phosphorylation during the differentiation of keratinocytes. Our observations suggest that HSP25 is involved in two steps of PAM212 keratinocyte differentiation. Shortly after the induction of differentiation, a transient hyperphosphorylation of HSP25 seems to be essential for the expression of differentiation markers. Later, the chaperone-active form of HSP25 is organized progressively into characteristic aggregates involved in the dynamics of keratin filament networks.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

Cold atmospheric plasma ameliorates imiquimod-induced psoriasiform dermatitis in mice by mediating antiproliferative effects

Psoriasis is a chronic hyperproliferative skin disease characterised by excessive growth of keratinocytes. Indeed, inducing keratinocyte apoptosis is a key mechanism responsible for psoriatic plaques clearance following some important existing therapies, which display pro-oxidant activity. Cold atmospheric plasma (CAP), acting as a tuneable source of reactive oxygen and nitrogen species (RONS), can controllably transfer RONS to the cellular environment, deliver antiproliferative RONS concentrations and exert antiproliferative and proapoptotic effects. This study was undertaken to evaluate the therapeutic potential of CAP in psoriasis. We used cell models of psoriasis-like inflammation by adding lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) to HaCaT keratinocytes. Indirect plasma, plasma-activated medium (PAM), was administered to HaCaT cells. Atmospheric pressure plasma jet (APPJ) was applied directly to imiquimod (IMQ)-induced psoriasiform dermatitis in mice. The results showed that PAM induced an increase in intracellular ROS and caused keratinocyte apoptosis. Moreover, cells under inflammation showed lesser viability and larger apoptosis rate. With repeated administration of APPJ, psoriasiform lesions showed ameliorated morphological manifestation and reduced epidermal proliferation. Overall, this study supports that CAP holds good potential in psoriasis treatment.