M-CSF-stimulated myeloid cells can convert into epithelial cells to participate in re-epithelialization and hair follicle regeneration during dermal wound healing.

Dermal wound healing is a complex process which requires the interaction of many cell types and mediators in a highly sophisticated temporal sequence. Myeloid cells which compose of a significant proportion of the inflammatory cells infiltrate to the to a wound site where they play important roles in clearance of damaged tissue and microorganisms. Myeloid cells have the capacity to be converted into fibroblast-like cells and endothelial cells during wound healing process. However, whether myeloid cells in wounds can convert into epithelial cells where they contribute to healing process is not clear. In this study, we performed double immunofluorescent staining with antibodies for hematopoietic cells and keratinocytes as well as cell tracing technique to investigate hematopoietic cell conversion. The result showed that during the healing process, some of the CD45-positive hematopoietic cells also expressed keratin 14, a marker for keratinocytes. Further, double immunofluorescent staining in dermal wounds, using CD11b and K14 antibodies indicated that CD11b-positive myeloid cells were the origin of newly generated epithelial cells. Through tracing injected labeled splenocyte-derived myeloid cells in skin, we confirmed that myeloid cells were able to convert into keratinocytes in repaired skin. Furthermore, our results from in vivo experiments provided new information on contribution of myeloid cells in hair follicle regeneration. In conclusion, this work highlights the myeloid cell contributions in wound repair and hair follicle regeneration through conversion of M-CSF-stimulated CD11b-positive myeloid cells into epithelial cells in a murine model.

Myeloid Adherent Cells Are Involved in Hair Loss in the Alopecia Areata Mouse Model.

Alopecia areata (AA), which is defined as an autoimmune hair loss disease, has a serious impact on the quality of life for patients with AA worldwide. In this study, to our knowledge, a previously unreported method of AA induction in C3H mice has been established and validated. Using this method, we showed that dermal injection of 1-3 million of a mixture of skin cells freshly isolated from AA-affected skin induces AA in more than 80% of healthy mice. Contrary to the previous protocol, the induction of AA by this approach does not need any surgical AA skin grafting, cell manipulation, or high number of activated T cells. We also showed that dermal injection of adherent myeloid cells (mainly CD11b+) in healthy mice is as potent as a mixture of none adherent CD3+ T cells and CD19+ B cells in the induction of AA. Interestingly, most of the mice (7 out of 8) that received non-adherent cells developed AA universalis, whereas most of the mice (5 out of 7) that received adherent cells developed patchy AA. Finally, we found a high number of stage-specific embryonic antigen-expressing cells whose expression in monocytes in an inflammatory disease causes the release of inflammatory cytokines, TNF-α and IL-1ß, from these cells in AA-affected skin.

Methotrexate modulates the expression of MMP-1 and type 1 collagen in dermal fibroblast.

Methotrexate (MTX), an anti-metabolite and anti-inflammatory drug, has been used to effectively manage and prevent keloids, but its mechanism(s) of action has not been elucidated. Our study sought to evaluate the effect of MTX on the production of key extra cellular matrix components, collagen, and matrix metalloproteinase-1 (MMP-1), produced by fibroblasts and involved in development of fibrosis. The proliferation and viability of cultured human dermal fibroblasts in response to different concentrations of MTX were determined using cell counting and MTT assay, respectively. Western blot analysis was used to determine the levels of both intracellular and secreted type 1 collagen and MMP-1. The results showed no significant changes in the proliferation of fibroblasts treated with 50 ng/ml of MTX as compared to that of control. Under the same experimental conditions, the level of secreted and intracellular type I collagen was markedly reduced and, conversely, the level of MMP-1 increased in treated neonatal, adult, and hypertrophic scar fibroblasts as compared with those of controls. The possible involvement of MTX-induced extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in MMP-1 production was also studied and the result showed an increase in phosphorylated ERK 1/2 in response to MTX treatment. In summary, the findings of this study revealed that MTX significantly reduced collagen production in different strains of fibroblasts derived from neonatal, adult, and hypertrophic scar tissues, while under the same experimental conditions, it increased the expression of MMP-1. As such, our findings validate and identify a potential mechanism through which MTX functions as an anti-fibrogenic factor in treating fibroproliferative disorders.

Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression.

Airway remodelling in asthma involves various mediators modulating the production/breakdown of collagen by lung fibroblasts. Matrix metalloproteinase-1 (MMP-1) plays an important role in collagen breakdown. We recently showed that epithelial cell-derived extracellular form of 14-3-3σ is an important inducer of MMP-1 expression in skin fibroblasts. Thus, we hypothesized that 14-3-3 proteins are important regulators of MMP-1 expression in the respiratory airway. We examined the presence of extracellular 14-3-3 proteins in conditioned media obtained from primary lung epithelial cells, A549 and HS24 cells, and their effect on MMP-1 expression by lung fibroblasts (IMR-90). In addition, we evaluated IMR-90 response to 14-3-3 proteins in the presence of transforming growth factor-ß(1) (TGF-ß(1)), a cytokine known to decrease MMP-1 expression by fibroblasts. Extracellular 14-3-3α/ß, but not -σ, is released by the human-derived lung epithelial cell lines, A549 and HS24. Unlike dermal fibroblasts, IMR-90 cells do not produce MMP-1 in response to 14-3-3σ. Conversely, MMP-1 production was induced following treatment of IMR-90 with recombinant or lung epithelial cell-derived 14-3-3α/ß. These findings were also confirmed using primary human bronchial epithelial cells and lung fibroblasts obtained from non-asthmatic patients. The MMP-1-inducing effect of 14-3-3α/ß on IMR-90 was not inhibited by TGF-ß(1). Lung epithelial cell-derived 14-3-3α/ß has a potent MMP-1-inducing effect on airway fibroblasts. Modulation of MMP-1 by 14-3-3α/ß, may be important in the alteration of collagenase production associated with airway remodelling in obstructive lung diseases. Our data indicate that 14-3-3 proteins may be potential targets for future therapeutic strategies aimed at modulating tissue remodelling in asthma.

14-3-3 sigma associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.

Matrix metalloproteinases (MMPs) are implicated in the degradation of the extracellular matrix during development and tissue repair, as well as in pathological conditions such as tumor invasion and fibrosis. MMP expression by stromal cells is partly regulated by signals from the neighboring epithelial cells. Keratinocyte-releasable 14-3-3sigma, or stratifin, acts as a potent MMP-1-stimulatory factor in fibroblasts. However, its mechanism of transmembrane signaling remains unknown. Ectodomain biotin labeling, serial affinity purification and mass spectroscopy analysis revealed that the stratifin associates with aminopeptidase N (APN), or CD13, at the cell surface. The transient knockdown of APN in fibroblasts eliminated the stratifin-mediated p38 MAP kinase activation and MMP-1 expression, implicating APN in a receptor-mediated transmembrane signaling event. Stratifin deletion studies implicated its C-terminus as a potential APN-binding site. Furthermore, the dephosphorylation of APN ectodomains reduced its binding affinity to the stratifin. The presence of a phosphorylated serine or threonine residue in APN has been implicated. Together, these findings provide evidence that APN is a novel cell surface receptor for stratifin and a potential target in the regulation of MMP-1 expression in epithelial-stromal cell communication.

Improvement of hypertrophic scarring by using topical anti-fibrogenic/anti-inflammatory factors in a rabbit ear model.

This study investigates the scar-reducing efficacy of topical application of stratifin and acetylsalicylic acid (ASA) in a rabbit ear model. A total of five New Zealand white rabbits with four wounds per ear were examined. Either recombinant stratifin (0.002%) or ASA (0.5%) incorporated in carboxymethyl cellulose gel was topically applied on each wound at postwounding Day 5. Scars were harvested at postwounding Day 28 for histological analysis. The wounds treated with stratifin and ASA showed 82 and 73% reduction in scar volume, respectively, compared with that of untreated controls. A reduction of 57 and 41% in total tissue cellularity along with 79 and 91% reduction in infiltrated CD3+ T cells were observed in response to treatment with stratifin and ASA, respectively, compared with those of untreated controls. Wounds treated with stratifin showed a 2.8-fold increase in matrix metalloproteinase-1 expression, which resulted in a 48% decrease in collagen density compared with those of untreated controls. Qualitative wound assessment showed a reduced hypertrophic scarring in stratifin and ASA-treated wounds when compared with the controls. This study showed that topical application of either stratifin or ASA-impregnated carboxymethyl cellulose gel reduced hypertrophic scar formation following dermal injuries in a rabbit ear fibrotic model.

Transdifferentiation of peripheral blood mononuclear cells into epithelial-like cells.

Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3varsigma for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3varsigma and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P

The role of stratifin in fibroblast-keratinocyte interaction.

Stratifin is a member of 14-3-3 protein family, a highly conserved group of proteins constituted by seven isoforms. They are involved in numerous crucial intracellular functions such as cell cycle and apoptosis, regulation of signal transduction pathways, cellular trafficking, cell proliferation and differentiation, cell survival, and protein folding and processing, among others. At epidermal level, stratifin (also called 14-3-3 sigma) has been described as molecule with relevant functions. For instance, this isoform is a marker associated with keratinocyte differentiation. In this maturation process, the presence of dominant negative molecules of p53 induces a "stemness condition" of keratinocyte precursor cells and suppression of stratifin expression. In addition, the recently described keratinocyte-releasable form of stratifin is involved in dermal fibroblast MMP-1 over-expression through c-Fos and c-Jun activity. This effect is mediated, at least in part, by p38 mitogen-activated protein kinase (MAPK). Other MMP family members such as stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), neutrophil collagenase (MMP-8), and membrane-type MMP-24 (MT5-MMP) are also up-regulated by stratifin. Within fibroproliferative disorder of skin, hypertrophic scar and keloids exhibit a high content of collagen, proteoglycans, and fibronectin. Thus, the MMP profile induced by stratifin is an interesting starting point to establish new therapeutic tools to control the process of wound healing. In this review, we will focus on site of synthesis and mode of action of stratifin in skin and wound healing.

14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes.

The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin alpha6, integrin beta4, or bullous pemphigoid antigen-1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14-3-3 sigma, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3sigma GST pull-down screening assay and showed that sigma (sigma) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3sigma revealed that only intact 14-3-3sigma molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 sigma is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 sigma interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3sigma isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.

Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts.

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.

Temperature-sensitive polymer-conjugated IFN-gamma induces the expression of IDO mRNA and activity by fibroblasts populated in collagen gel (FPCG).

Indoleamine 2,3-dioxygenase (IDO) is an intracellular tryptophan-catabolizing enzyme possessing various immunosuppressive properties. Here, we report the use of this enzyme to suppress the proliferation of peripheral blood mononuclear cells (PBMC) co-cultured with IDO-expressing fibroblasts of an allogeneic skin substitute in vitro. Fetal foreskin fibroblasts populated within collagen gel (FPCG) were treated with interferon-gamma (IFN-gamma) conjugated with a temperature-sensitive polymer to induce the expression of IDO mRNA and protein. SDS-PAGE showed successful conjugation of IFN-gamma with the temperature-sensitive polymer. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by the measurement of kynurenine levels. The results of Northern blot analysis showed an induction of IDO mRNA expression when treated with polymer-conjugated IFN-gamma. Kynurenine levels, as a measure of IDO bioactivity, were significantly higher in IFN-gamma-treated fibroblasts than in controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA in FPCG treated with polymer-conjugated IFN-gamma was significantly longer than in those treated with free (non-conjugated) IFN-gamma (P < 0.001). IFN-gamma radiolabeling showed a prolonged retention of IFN-gamma within collagen gel in its polymer-conjugated form, compared to its free form. Presence of IDO protein in FPCG was demonstrated by Western analysis even 16 days after removal of the conditioned medium (containing released IFN-gamma). To demonstrate the immunosuppressive effects of IDO on the proliferation of PBMC, IDO-expressing FPCG treated with polymer-conjugated IFN-gamma were co-cultured with PBMC for a period of 5 days. The results showed a significant reduction in proliferation of PBMC co-cultured with IFN-gamma-treated IDO-expressing fibroblasts, compared to those co-cultured with non-IDO-expressing fibroblasts (P < 0.001). The addition of an IDO inhibitor (1-methyl-D-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation. In conclusion, IDO expression in FPCG suppresses the proliferation of immune cells in vitro. The use of a temperature-sensitive polymer further prolongs the effect of IFN-gamma on the expression of IDO. Therefore, modulating IDO levels in situ might be an alternative for prolonging the survival of skin allografts.

Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor.

As a possible way of making a non-rejectable skin substitute, here, we ask the question of whether the expression of indoleamine 2,3-dioxygenase (IDO) selectively suppresses immune, but skin, cell proliferation. To address this question, a series of experiments in which adenovirus (Ad-IDO) infected IDO expressing dermal fibroblasts were co-cultured with different types of immune cells were carried out. The immune cells were then harvested and evaluated for propidium iodide (PI) positive cells by FACS analysis. TUNEL assay was also carried out to determine the apoptotic status of these cells. The results showed that the expression of IDO in dermal fibroblasts significantly induces apoptotic death of PBMC, CD4(+)-, CD8(+)- and B cell-riched primary lymphocytes, Jurkat cells, and THP-1 cells. IDO-mediated damage of immune cells was restored by an addition of tryptophan and IDO inhibitor. Using the same approaches, we also demonstrated that skin cells and endothelial cells are remarkably resistant to tryptophan-deficient environment. Furthermore, no significant difference in cell proliferation between Ad-GFP (control)- and Ad-IDO-GFP-infected either keratinocytes or fibroblasts, was found. The results of this study, therefore, suggest that the expression of IDO by dermal fibroblasts mediates immune cell damage and this may shed a new light toward developing a non-rejectable skin substitute in the future.

Infection of placental trophoblasts by Toxoplasma gondii.

How the intracellular parasite Toxoplasma gondii causes placental inflammation and infects the fetus is unknown. By use of a culture model of primary human trophoblasts, we examined the consequences of infection by a virulent strain of T. gondii. Infection fractions (parasitophorous vacuoles per trophoblast nuclei) < or =0.9 were observed 1 day after challenge at an inoculum ratio of T. gondii to nuclei of 10. The culture content of infectious T. gondii increased 45-fold in 48 h. Two days after infection, almost 30% of trophoblast nuclei became apoptotic, and 30%-35% of nuclei were lost. Almost 90% of apoptotic nuclei were not adjacent to a parasitophorous vacuole, suggesting infection protected against apoptosis. However, there was no T. gondii-dependent accumulation of putative cytotoxic factors, such as tumor necrosis factor-alpha, that could mediate paracrine killing. Both mature and immature trophoblasts can be productively infected, and uninfected, but not infected, cells undergo apoptosis.

Selective reovirus killing of bladder cancer in a co-culture spheroid model.

Up to 50% of the transitional cell carcinomas (TCC) express an activated EGF pathway involving MAP/MEK and RAF kinase thus providing a novel means to selectively eliminate transformed cells expressing such proteins. This EGF pathway expression phenotype was also confirmed in our MGH-U3 and room temperature-112 human TCC cell lines, which makes them a suitable model target for the reovirus oncolysis. We report here on an in vitro assay of co-culture spheroids using either human or rat TCC cells with their corresponding fibroblasts to examine the potential of viral selective lysis for TCC. Reovirus, a respiratory enteric orphan virus, which mammals are exposed to early in life, was used in this study. Selective killing of transformed versus normal cells was assayed by time-lapse photography, vital dye staining, immunohistochemistry, and MTT assay. In this in vitro bladder cancer model, reovirus selectively destroyed the transformed cells by lysis or induction of apoptosis. Based on these findings we have initiated an in vivo pre-clinical study on intravesical administration of reovirus in an animal model to further explore the effect of reovirus-mediated oncolysis of TCC.