From the bench to the clinic: New aspects on immunoregulation by vitamin D analogs.

The skin is a vital organ that plays a crucial role in defending us from pathogens. Multiple players from the innate and adaptive immune system are involved, such as neutrophils, dendritic cells, lymphocytes and antimicrobial peptides. Chronic inflammatory skin diseases can be mediated by inflammatory T cells and their interactions with other cells in the skin. Vitamin D is generated in the skin upon sun exposure and has a variety of effects. Vitamin D and its analogs have been used with success in treating mild to moderate T cell-mediated skin diseases, but how they mediate the beneficial effects is not well understood. In the recent years, emerging evidence is rising that vitamin D analogs and its modulation on the immune system plays a major role. It has been shown that vitamin D analogs can induce the generation of regulatory T cells, which are able to suppress proliferation and alter the function of inflammatory T cells. This may help explain the therapeutic effects that are observed and at the same time give hope that in combination with other therapy or used alone, vitamin D analogs may be helpful when treating more severe forms of the diseases.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Improving topical treatments for skin diseases.

Skin diseases and skin problems affect millions of people every day. Skin lesions are often difficult to treat locally and much is to be gained by developing more targeted treatments to avoid the use of systemic immunosuppressant drugs. Recent data on the function of microenvironmental factors could help improve such therapies. Vitamins A and D and their metabolites have strong effects on both innate and adaptive immune responses, and can suppress inflammation, lymphocyte infiltration and function, as well as the ability to increase regulatory tasks within the skin. Combinatorial therapy that includes these metabolites, cytokines, chemokines or other agents could help to alter the local microenvironment and sway immune responses as needed, depending on the origin of the skin inflammation. Here, I argue that microenvironmental factors might be used to improve topical treatments in chronic inflammatory skin diseases such as psoriasis and atopic dermatitis, where T cells play a pathogenic role.

Fundamental questions to sun protection: A continuous education symposium on vitamin D, immune system and sun protection at the University of Zürich.

Since exposure to sunlight is a main factor in the development of non-melanoma skin cancer and there are associations between malignant melanoma and short-term intense ultraviolet (UV) exposure, particularly burning in childhood, strict protection from UV-radiation is recommended. However, up to 90% of all requisite vitamin D has to be formed within the skin through the action of the sun-a serious problem, for a connection between vitamin D deficiency, demonstrated in epidemiological studies, and various types of cancer and other diseases has been confirmed. A UVB-triggered skin autonomous vitamin D(3) synthesis pathway has recently been described, producing the active Vitamin D metabolite calcitriol. This cutaneous vitamin D(3) pathway is unique. Keratinocytes and dendritic cells can convert vitamin D to calcitriol. Cutaneous T cells activated in the presence of calcitriol express the chemokine receptor CCR10 attracting them to the chemokine CCL27 that keratinocytes express selectively in the epidermis, and migrate from dermal layers of the skin to the epidermis under UV radiation. Thus, calcitriol has endocrine roles beyond its calciotropic action, including cell growth and cancer prevention. Therefore, strict sun protection procedures to prevent skin cancer may induce the risk of vitamin D deficiency. As there is evidence that the protective effect of less intense solar radiation can outweigh its mutagenic effect, better balanced approaches to sun protection should be sought.

Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking.

Lymphocytes are imprinted during activation with trafficking programs (combinations of adhesion and chemoattractant receptors) that target their migration to specific tissues and microenvironments. Cytokines contribute, but, for gut and skin, evolution has cleverly adapted external cues from food (vitamin A) and sunlight (ultraviolet-induced vitamin D3) to imprint lymphocyte homing to the small intestines and T cell migration into the epidermis. Dendritic cells are essential: they process the vitamins to their active metabolites (retinoic acid and 1,25(OH)(2)D3) for presentation with antigen to lymphocytes, and they help export environmental cues through lymphatics to draining lymph nodes, to program the trafficking and effector functions of naive T and B cells.

DCs metabolize sunlight-induced vitamin D3 to 'program' T cell attraction to the epidermal chemokine CCL27.

During adaptive immune responses, dendritic cells activate T cells and endow them with specific homing properties. Mechanisms that 'imprint' specific tropisms, however, are not well defined. We show here that 1,25(OH)(2)D(3), the active form of vitamin D3, signaled T cells to express CC chemokine receptor 10, which enabled them to migrate to the skin-specific chemokine CCL27 secreted by keratinocytes of the epidermis. In contrast, 1,25(OH)(2)D(3) suppressed the gut-homing receptors alpha4beta7 and CCR9. Vitamin D3, the inactive prohormone naturally generated in the skin by exposure to the sun, was processed by dendritic cells and T cells to the active metabolite, providing a mechanism for the local regulation of T cell 'epidermotropism'. Our findings support a model in which dendritic cells process and 'interpret' locally produced metabolites to 'program' T cell homing and microenvironmental positioning.

Narrowband-UVB irradiation decreases the production of pro-inflammatory cytokines by stimulated T cells.

Narrow-band ultraviolet B (UVB) phototherapy is an effective treatment for psoriasis. Owing to its limited penetration, the direct effects of UVB are mostly restricted to cells residing in the epidermis and papillary dermis, and are associated with epidermal depletion of Langerhans' cells (LC) and T cells. It has been argued that the depletion of the skin-resident T-cell population may be due to a combination of UVB-induced apoptosis and decreased recruitment from the blood due to lower expression of the required adhesion and homing molecules. We have previously demonstrated that UVB treatment can alter the expression of adhesion molecules by blood lymphocytes, and as these can be influenced by cytokines, the aim of this study was to investigate whether UVB irradiation can also influence the cytokine production of circulating T cells. Four patients with active chronic plaque psoriasis were treated daily with narrow-band 312 nm UVB irradiation and blood samples obtained before treatment and weekly thereafter for 2 weeks. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with a streptococcal superantigen or a conventional streptococcal antigen preparation, and cell culture supernatants were assayed for various cytokines. When stimulated with the superantigen, PBMCs from UVB-treated psoriasis patients secreted greater amounts of the anti-inflammatory cytokine IL-10, and showed markedly decreased production of IL-1beta, IL-2, IL-5 and IL-6 compared to the pre-treatment values; the production of IFN-gamma, IL-8 and IL-12p70 were also decreased but did not reach statistical significance. Thus, the combination of UVB-induced apoptosis, increased secretion of anti-inflammatory cytokines and decreased trafficking to the skin may help to explain the beneficial effects of UVB treatment on psoriasis and why disease remission can sometimes be sustained for a prolonged period.

The anti-inflammatory action of methotrexate is not mediated by lymphocyte apoptosis, but by the suppression of activation and adhesion molecules.

Low-dose methotrexate (MTX) is an established and highly effective treatment for severe psoriasis and rheumatoid arthritis; however, its mechanism of action remains unclear. We investigated the effects of low-dose MTX on antigen-stimulated peripheral blood mononuclear cells and explored through which cellular pathways these effects are mediated. We show that MTX caused a dose-dependent suppression of T cell activation and adhesion molecule expression, and this was not due to lymphocyte apoptosis. The suppression of intercellular adhesion molecule (ICAM)-1 was adenosine and folate-dependent, while MTX suppression of the skin-homing cutaneous lymphocyte-associated antigen (CLA) was adenosine-independent. The effect of MTX on CLA, but not ICAM-1, required the constant presence of MTX in cultures. Thus, the suppression of T cell activation and T cell adhesion molecule expression, rather than apoptosis, mediated in part by adenosine or polyglutamated MTX or both, are important mechanisms in the anti-inflammatory action of MTX.

Methotrexate markedly reduces the expression of vascular E-selectin, cutaneous lymphocyte-associated antigen and the numbers of mononuclear leucocytes in psoriatic skin.

A positive correlation between disease severity and the frequency of cutaneous lymphocyte-associated antigen (CLA)-positive T cells in the blood of untreated patients with psoriasis has been previously observed. A dose-dependent inverse relationship between disease severity and the frequency of circulating CLA(+) T cells in psoriasis patients on methotrexate (MTX) treatment is reported. Circulating T cells from a patient with psoriasis were monitored for CLA expression on a daily basis for 5 weeks. A decrease in the intensity and frequency of CLA(+) mononuclear leucocytes was consistently observed in the blood during the first 3-4 days after each MTX intake, but the CLA expression increased thereafter until the next weekly dose was taken. The MTX treatment of this patient was then discontinued for 16 days, and a marked subjective exacerbation was reported within 9 days, which was confirmed objectively (laser Doppler perfusion imaging) after 11 and 16 days. Biopsies taken 4 days after the last MTX intake showed only a few mononuclear leucocytes in lesional skin, but the exacerbation coincided with a marked increase in CLA expression by mononuclear blood leucocytes, followed by an increase in endothelial E-selectin and a striking influx of CLA(+) mononuclear cells into lesional skin. Conversely, a clinical improvement after the patient resumed the MTX treatment was associated with reduction in CLA expression by mononuclear cells in the blood, downregulation of endothelial E-selectin and an approximate threefold decrease in mononuclear leucocyte infiltration of lesional skin. No MTX-associated changes were detected in the expression of very late antigen-4, vascular cell-adhesion molecule-1 nor the late activation marker CD25. It is concluded that MTX decreases the expression of CLA and E-selectin and that this may be a major mechanism for the therapeutic effect of MTX on psoriatic skin lesions.

Differential effects of interleukin 12 and interleukin 10 on superantigen-induced expression of cutaneous lymphocyte-associated antigen (CLA) and alphaEbeta7 integrin (CD103) by CD8+ T cells.

At both cutaneous and mucosal sites, interleukin (IL)-10, IL-12 and transforming growth factor (TGF)-beta are important regulators of chronic inflammatory disease, where cutaneous lymphocyte-associated antigen (CLA) and alphaE integrin (CD103) may be expressed. Stimulation with streptococcal pyrogenic exotoxin C (SpeC) increased the expression of CD103 by CD8+ but not CD4+ T cells. While adding IL-12 augmented the expression of CLA, superantigen-induced expression of CD103 was markedly suppressed by IL-12, which could be reversed by TGF-beta. Antibodies against TGF-beta inhibited, and a combination of anti-TGF-beta and IL-12 completely abrogated the induced CD103 expression. IL-10 strongly decreased the frequency of CLA+ and although not increasing the frequency of CD103+CD8+ T cells, the amount of CD103 expressed per cell was markedly increased. Thus, the expression of CLA and CD103 may be antagonistically regulated by IL-10 and IL-12 and the balance between these cytokines could influence the T cell migration of inflammatory cells into epithelial tissues.