CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> regulatory T cells in human lupus erythematosus

Natural CD4⁺CD25⁺ regulatory T cells (T_reg) show a potent immunosuppressive function and contribute to immunologic self-tolerance by suppressing potentially auto-reactive T cells. Depletion of these cells leads to the induction of severe autoimmune diseases in animal models; more recently, several studies have also reported an impairment of T_reg number and/or function in various human autoimmune diseases. For example, aberrant numbers of circulating CD4⁺CD25⁺ T_reg have been seen in patients with type I diabetes, mycosis fungoides, graft-versus-host-reaction, and rheumatoid arthritis. Moreover, increased numbers of functionally active CD4⁺CD25⁺ T_reg have been detected in the synovial fluid of patients with rheumatoid arthritis. In systemic lupus erythematosus (SLE), conflicting data on the role of CD4⁺CD25⁺ T_reg in human autoimmune diseases have been presented in the literature. Decreased numbers of peripheral blood T_reg have been reported by most studies on SLE patients with active disease, but non-impaired or even increased CD4⁺CD25⁺ T_reg numbers have also been described. In addition, both deficient and normal suppressive capacity of isolated T_reg have been observed in SLE. Analysis of CD4⁺FoxP3⁺ T_reg in skin lesions of patients with a primarily cutaneous manifestation of the disease showed a significant reduction in cell numbers as compared to other inflammatory skin diseases, suggesting the importance of analyzing T_reg numbers in the affected tissue. In this review, we discuss the role of CD4⁺CD25⁺ T_reg in autoimmunity and recent published data on SLE. Furthermore, we highlight the need for additional studies that address specific gaps of knowledge regarding the pathophysiological mechanisms as well as the identification of future therapeutic strategies for autoimmune diseases.     

In situ depletion of CD4<Superscript>+</Superscript> T cells in human skin by Zanolimumab

CD4⁺ T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4⁺ T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, κ) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4⁺, but not CD8⁺ CD3⁺ T cells. The capacity of Zanolimumab to deplete the CD4⁺ T cells in the skin may be of importance in diseases where CD4⁺ T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.     

Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8<Superscript>+</Superscript>CLA<Superscript>+</Superscript> T cells

Background

Recent studies have shown that vitiligo is a T-cell mediated autoimmune disease. Skin-homing cytotoxic T lymphocytes expressing cutaneous lymphocyte-associated antigen (CLA) have been suggested to be responsible for the destruction of melanocytes in vitiligo. An aberration in the suppressive function of regulatory T cells (Tregs) has been reported in vitiligo patients. However, whether the weakened suppressive ability of the Tregs contributes to hyper-activated skin homing CD8⁺CLA⁺ T cells remains to be determined.

Objectives

To investigate the inhibition of circulating Tregs on the proliferation of autologous CD8⁺CLA⁺ T cells in non-segmental vitiligo patients.

Methods

CD8⁺CLA⁺ T cells and Tregs were obtained from the peripheral blood of 13 non-segmental vitiligo patients and 7 controls. The proliferative responses of CD8⁺CLA⁺ T cells were assessed in the absence or presence of autologous Tregs, and the levels of Transforming Growth Factor β1(TGF-β1) and IL-10 in culture supernatants were detected by enzyme-linked immunosorbent assay.

Results

The proliferative responses of circulating CD8⁺CLA⁺ T cells in the presence of Tregs were significantly higher in the active vitiligo than in the stable vitiligo and control groups. Tregs from active vitiligo patients exhibited a lower inhibitory effect on proliferation of CD8⁺CLA⁺ T cells. The levels of TGF-β1 produced by Tregs were significantly lower in active vitiligo than other groups and anti-TGF-β1 antibodies could abrogate the suppressive function of Tregs.

Conclusions

The functional activity of Tregs is compromised in active vitiligo patients. TGF-β1 plays an important role in the autoimmune mechanism of the disease.

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Administration of substance P during a primary immune response amplifies the secondary immune response via a long-lasting effect on CD8<Superscript>+</Superscript> T lymphocytes

Atopic dermatitis can be exacerbated or induced by scratching or psychological stress; both cause the release of substance P (SP) from sensory nerves. Therefore, SP may have an etiological role in mechanisms underlying AD. Here, we show that administration of SP during the primary immune response (PIR) imprinted long-lasting pro-inflammatory immunity, resulting in exacerbation of the secondary immune response (SIR) in the absence of further SP. Five days after sensitization with dinitrofluorobenzene (DNFB), challenge with DNFB together with SP (“SP-Group”) resulted in an increased PIR (as evaluated by ear swelling and granulocyte infiltration) compared to DNFB only (“Control-Group”). On day 26, after inflammation completely subsided, a second challenge with DNFB only (without SP) caused an increased SIR in the “SP-Group” compared to controls. Pretreatment on day 5 with spantide, an SP receptor antagonist, prevented increased ear swelling in the “SP-Group” not only on day 5 (PIR) but also on day 26 (SIR). In contrast, spantide treatment on day 26 did not affect the SIR. Adoptive transfer experiments suggested that CD8⁺ T cells were involved in mediating enhanced SIR in animals pretreated on day 5 with SP. The present study offers a novel experimental approach to an uninvestigated facet of the pro-inflammatory effect of SP, i.e., exacerbation of inflammation via a long-term and indirect influence on CD8⁺ T lymphocytes.     

<Superscript>18</Superscript>F-fluorodeoxyglucose-positron emission tomography is more sensitive than computed tomography in initial staging of patients with an anaplastic T-cell lymphoma first presenting in the skin

Background

The role of ¹⁸F-fluorodeoxyglucose-positron emission tomography (FDG-PET) in the evaluation of anaplastic large-cell lymphoma (ALCL) first presenting in the skin is not well established, while computed tomography (CT) is used as a standard procedure.

Objectives

The aim of this study was to evaluate the use of FDG-PET versus CT at initial staging of ALCL first presenting in the skin.

Materials & methods

Eleven cases of ALCL first presenting in the skin who underwent both FDG-PET and CT were retrospectively analysed. There were six males and five females, with a mean age of 59.7 years. Results of FDG-PET were compared with those of CT. Biopsy results of lesions served as a reference for the accuracy of PET and CT in the evaluation of local and metastatic lesions.

Results

In seven cases (64%), imaging revealed extracutaneous ALCL. FDG-PET results were concordant with CT results in five cases (45%); in four of these cases, FDG-PET was negative, consistent with CT, and one case had cutaneous and extracutaneous lesions detectable on both CT and FDG-PET. FDG-PET and CT were discordant in six cases (55%). Among these six cases, FDG-PET revealed extracutaneous lesions undetected on CT which consequently influenced the therapeutic decision in five cases (45%).

Conclusion

The sensitivity of CT and FDG-PET was 18% and 64%, respectively.

     

Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma

Abstract:  Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor β. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.     

Lymphocytes and macrophages of the epidermis and dermis in lesional psoriatic skin,but not epidermal Langerhans cells,are depleted by treatment with cyclosporin A

Summary Since cyclosporin A (CsA) is an immuno-suppressive agent, its beneficial effect in psoriasis suggests that immune cells may play a role in the pathogenesis and resolution of psoriasis. To determine early effects of CsA in psoriasis, we quantitated immune cells using double immunofluorescence microscopy on biopsy specimens obtained prior to therapy and after 3,7, and 14 days of CsA therapy. CsA therapy resulted in significant reductions in the absolute number of immune cells (including T cells, monocytes/macrophages, and antigen presenting cells) contained within psoriatic skin. The effect was rapid, with over one-half of the reduction in the density of HLe1⁺ (human leukocyte antigen-1 positive or bone marrow derived) cells, including T cells, activated T cells, monocytes, and Langerhans cells (LCs), occurring within 3 days. Despite the overall reduction in the numbers of immunocytes in the skin, the proportion of T cells, Langerhans cells, and monocytes in relation to the total number of immune cells was unchanged with therapy, reflecting equally proportional losses of each subtype. Dermal CD1⁺DR⁺ cells (putative Langerhans cells), which are not found in normal skin but are present in lesional psoriasis skin, were virtually cleared from the papillary dermis after CsA therapy. Although absolute numbers of epidermal Langerhans cells, defined as cells expressing both CD1 (T6) and DR molecules (CD1⁺DR⁺), were also reduced after CsA, epidermal non-Langerhans CD1^-DR⁺ cells (macrophages, activated T cells, DR^- keratinocytes) demonstrated a proportionally greater decrease, with the ratio of CD1⁺DR⁺ Langerhans cells/non-Langerhans CD1^-DR⁺ epidermal cells changing from a mean of 0.82 at baseline to 1.92 at day 14. Thus, early in the course of therapy, CsA appears to be effective at clearing CD1^-DR⁺ cells while leaving LC relatively intact in the epidermis.This work was supported in part by the Babcock Foundation     

Balance of Treg vs. T‐helper cells in the transition from symptomless to lesional psoriatic skin

Background In the pathogenesis of psoriasis, proinflammatory T cells are strongly involved in the inflammatory process, where regulatory T‐cell (Treg) function is impaired. Objectives As effective Treg function is associated with a numerical balance between Treg and effector T cells, we wondered whether Treg/T‐helper cell ratios may be associated with certain stages of the inflammatory process. We opted for the margin zone model as a dynamic approach. Methods From nine patients with chronic plaque psoriasis, 3‐mm punch biopsies were obtained from the centre and margin of the lesion, perilesional skin and distant uninvolved skin. Skin biopsies of 10 healthy volunteers were included as a control. Samples were analysed using immunohistochemistry and immunofluorescence. Results In the transition from symptomless to lesional skin, a significant increase of CD3+, CD4+ and Foxp3+ cells was found. In seven of nine patients the ratio of Treg (Foxp3+) vs. CD4+ T cells was higher in the distant uninvolved skin than in the perilesional and lesional skin. Interestingly, the Foxp3/CD4 ratio in the distant uninvolved skin was even higher than in the skin of healthy controls. Notably, we found that most of the interleukin (IL)‐17 expression was not related to CD4+ cells, but to mast cells. Conclusions The relatively high Foxp3/CD4 ratio in symptomless skin of patients with psoriasis suggests an active immune controlling mechanism distant from the psoriatic plaque. In the margin and centre of the plaque the ratio appears skewed towards effector cells associated with inflammation. IL‐17, an important driver of the psoriatic process, is mostly related to mast cells, and only sporadically to T cells.     

Increased expression of the natural killer cell inhibitory receptor CD94/NKG2A and CD158b on circulating and lesional T cells in patients with chronic plaque psoriasis

BACKGROUND: Psoriasis is a common inflammatory cutaneous disorder characterized by activated T-cell infiltration. T lymphocytes bearing natural killer cell receptors (NKRs) have been suggested to play an important role in the pathogenesis of psoriasis. However, the expression pattern of activating and inhibitory NKRs on T lymphocytes from psoriatic patients and its significance in psoriasis needs further study. OBJECTIVES: To investigate the pathogenesis of NKR-expressing T cells in psoriasis. MATERIALS AND METHODS: Thirty patients with chronic plaque psoriasis and 20 healthy controls were enrolled in this study. The immunophenotypic profiles of NKRs, including CD56, CD16 (activating NKRs), CD158a, CD158b, CD94 and NKG2A (inhibitory NKRs), were analysed in peripheral blood T lymphocytes, as well as psoriatic lesional infiltrating T cells, by triple-fluorescence flow cytometry. RESULTS: A significant increase of inhibitory CD8+ CD158b+, CD4 CD8 CD158b+ and CD8+ CD94/NKG2A+ T cells was found in the peripheral blood of patients with psoriasis when compared with controls. Tissue-infiltrating T lymphocytes expressing inhibitory receptors CD158b, CD94 and NKG2A were found in psoriatic lesions. There was a significant positive correlation between the increased percentage of circulating CD8+ CD94/NKG2A+ T cells and the Psoriasis Area and Severity Index. CONCLUSIONS: In the present study, we demonstrated increased proportions of particular subsets of inhibitory CD158b+ and/or CD94/NKG2A+ T cells in patients with psoriasis. The elevation of these inhibitory NKR-expressing T cells was correlated with disease severity, which may signify the possibility of chronic antigen-driven stimulation and dysregulated cytokine production in the pathogenesis of psoriasis.     

槲皮黄酮通过Sphk2抑制人皮肤鳞状细胞癌A431细胞的增殖

目的：研究槲皮黄酮（Quercetin, Qu）对人皮肤鳞状细胞癌A431细胞增殖活性的影响。方法：采用CCK8法、Western blot实验和流式细胞术检测不同浓度下（10、50、100、200 μg/mL）A431细胞活力、增殖及凋亡相关指标;Western blot检测Qu对A431细胞中Sphk2信号通路的影响。结果：10、50、100、200 μg/mL Qu作用96 h后,细胞OD值分别为（0.654±0.098）、（0.527±0.089）、（0.412±0.076）、（0.309±0.054）;细胞活力分别为（81.3±4.6）%、（56.3±4.9）%、（37.4±4.2）%、（19.4±3.6）%;细胞凋亡率分别为（6.43±1.09）%、（14.77±1.26）%、（21.30±1.36）%、(29.84±1.26)%。Qu呈浓度依赖性的抑制细胞中Sphk2蛋白表达,且Qu+Sphk2 mimics组细胞活力明显高于于Qu,细胞凋亡率明显低于Qu组(Ps<0.05)。结论：Qu可能通过抑制Sphk2信号通路降低人皮肤鳞状细胞癌细胞的增殖活性。     

The influence of sun exposure on the DNA methylation status of <Emphasis Type="Italic">MMP9</Emphasis>, <Emphasis Type="Italic">miR-137</Emphasis>, <Emphasis Type="Italic">KRT14</Emphasis> and <Emphasis Type="Italic">KRT19</Emphasis> genes in human skin

Background

Studies have shown that a variety of environmental factors and habits are associated with epigenetic changes. In addition, various genes are also found to respond to UV radiation.

Objectives

The aim of this study was to investigate the sun exposure influence on the DNA methylation profile on the matrix metalloprotease-9 (MMP9), microRNA 137 (miR-137), cytokeratin 14 (KRT14) and 19 (KRT19) genes of skin cells of subjects with no history of skin diseases.

Methods

Skin biopsies (5mm) were obtained using a punch technique on sun-exposed (outer forearm) and sun-protected areas (inner arm) from 30 corpses from the Brazilian Service of Death Investigation. Skin types were ranked according to Fitzpatrick’s criteria. Genomic DNA was extracted and a DNA methylation analysis was performed using Methylation Specific PCR (MSP) or Methylation-Sensitive Restriction Enzymes (MSRE) of sun-exposed and sun-protected skin areas.

Results

No differences were found among the areas (p>0.05; McNemar), with the partially methylated condition found to be a common event in skin for both MMP9 and miR-137 genes and the methylated condition for both KRT14 and KRT19 genes. Additional analysis showed no differences in the methylation status when age, gender and skin type were considered, however, the methylation status of miR-137 gene seems to be genderrelated.

Conclusions

We conclude that sun exposure does not induce changes in the DNA methylation status in MMP9, miR-137, KRT14 and KRT19 genes.

     

Production and pharmacologic modulation of the granulocyte-associated allergic responses to ovalbumin in murine skin models induced by injecting ovalbumin-specific Th1 or Th2 cells

Because interferon-gamma, interleukin-4, and interleukin-5 have been identified at the mRNA and protein levels in the lesional skin of patients with atopic dermatitis, we investigated the roles played by granulocytes as effector cells in allergic inflammation by using two unique murine skin models. In vitro generated Th1 and Th2 cells from na?ve splenocytes of antiovalbumin T cell receptor transgenic BALB/C mice were adoptively transferred with ovalbumin into the ear pinnae or air-pouches produced in the back skin of na?ve, nontransgenic BALB/C mice. The injection of Th1 cells with ovalbumin induced delayed type ear swelling that peaked at 48 h, whereas that of Th2 resulted in ear swelling that peaked at a much earlier time, 24 h. Histologic study of the swollen ear skin and granulocytes recruited into the air-pouch demonstrated that, although the Th1-induced inflammation caused a neutrophil-predominant infiltrate with few eosinophils, larger numbers of eosinophils accumulated in the Th2-induced inflammation. Using these murine models, we further evaluated the effects of drugs used for the treatment of atopic diseases. The results showed that FK506 administration could effectively reduce skin inflammation induced by either Th cells. Interestingly, the neutrophil elastase inhibitor ONO-6818 efficiently inhibited Th1-induced inflammation. In contrast, a leukotriene receptor antagonist, ONO-1078, specifically suppressed Th2-induced inflammation. We also found that each ONO drug exerted direct influence on specified granulocytes, as neither affected in vitro production of relevant Th cytokines. Thus, we succeeded in developing animal skin inflammation models in which we can evaluate the contribution of protein antigen-specific Th1 or Th2 cells through the action of granulocytic effector cells.     

Expression of Foxp3+CD4+CD25+ regulatory T cells and Th1/Th2, Tc1/Tc2 profiles in the peripheral blood of patients with condyloma acuminatum

Background.  Condyloma acuminatum (CA) is a disease that appears as proliferative lesions of the genital epithelium caused by human papillomavirus (HPV) infection. The balance between type 1 and type 2 T-cell subsets in patients with CA is thought to modulate antiviral immunity. CD4+CD25+ regulatory T cells (Tregs) inhibit proliferation and cytokine production by both T-helper (Th)1 and Th2 cells and reversibly suppress CTL-mediated immunity. A better understanding of the mechanisms of T-cell regulation in CA might help in developing more effective therapeutic strategies.
Objective.  To evaluate the balance of Th1/Th2 and Tc1/Tc2 and to assess their correlation with changes in number of Tregs in CA.
Methods.  The percentage of Th1, Th2, Tc1, Tc2 and Tregs were detected by flow cytometry after intracellular staining for cytokines (interferon-γ and interleukin-4) and Foxp3 of T lymphocytes in the peripheral blood of 30 patients and 20 healthy volunteers.
Results.  Patients with CA showed a decreased proportion of Th1 and Tc1 cells and a decreased ratio of Th1/Th2 and Tc1/Tc2. In particular, strikingly decreased ratios of Th1/Th2 were found in 15 patients with relapsed CA ( P  < 0.01). The mean ± SD number of Foxp3+CD4+CD25+ Tregs increased significantly in patients with CA (3.37 ± 1.03%) and patients with relapsed CA (4.68 ± 1.17%) compared with healthy controls (1.18 ± 0.53%) ( P  < 0.001).
Conclusion.  Tregs appear to downregulate cytokine expression in both Tc1 and Th1 subsets of effector T cells, which may be responsible for antivirus responses.     

<Emphasis Type="Italic">N</Emphasis>-Linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.