Modulation of MHC class II+ Langerhans cell numbers in corticosteroid treated epidermis by GM-CSF in combination with TNF-α

Abstract It is important to understand how dendritic cells (DC) are recruited, maintained and stimulated to migrate from tissues to lymph nodes. This is because DC are potent initiators of primary immune responses and candidates for vaccine development. Identification of factors which could lead to increased numbers of DC in tissues could affect immune responses by modulating their interaction with antigen which penetrates the tissue. To identify cytokines which could increase DC in tissues we tested the ability of GM-CSF, TNF-α and IL-6 to partially prevent steroid depletion of Langerhans cells (LC) from the epidermis. Cytokines diluted in serum-containing medium were compared with cytokines diluted in albumin-containing, serum-free medium in order to determine a minimum combination of cytokines required to increase LC and the effect of serum on the LC-increasing activity of cytokines. In the presence of serum, GM-CSF or TNF-α could increase LC frequency compared to the control; but in the absence of serum neither of these cytokines were effective unless they were combined with each other. In the presence of serum the combination of GM-CSF with TNF-α was ineffective. The data support the hypotheses that GM-CSF and TNF-α are both important in regulating LC numbers in the epidermis in vivo. Serum may modulate how each of these cytokines, separately or in combination, affect LC frequency in the epidermis–GM-CSF and TNF-α separately probably interact with other factors present in serum to increase LC frequency, whereas in combination it is possible that these separate effects are cancelled in the presence of serum. TNF-α and GM-CSF together, in the absence of serum, form one combination of a minimum number of cytokines which can regulate LC frequency in the epidermis; and IL-6 alone, or in combination with GM-CSF, does not increase LC frequency.     

T6+ and HLA-DR+ cell numbers in epidermis of immunosuppressed renal transplant recipients

The increased susceptibility of the skin of chronically immunosuppressed individuals to viral infections and sunlight-induced malignancies suggests specific drug-induced, dysfunction of local immune mechanisms within the sun-exposed skin of these individuals. To help understand the effect of immunosuppressive therapy alone in the absence of ultraviolet light on the immune system of skin, biopsies were collected from non-sun-exposed buttock skin of control, healthy volunteers and kidney transplant recipients immunosuppressed with either azathioprine/prednisone or cyclosporin A/prednisone and examined for incidences of T6+, and HLA-DR+ cells. No significant differences in the incidences of these 2 cell types were found (a) between control individuals and transplants recipients, (b) between transplant recipients receiving either of the immunosuppressive drug regimes, or (c) between transplant recipients who either had or had not developed skin cancer.     

Morphological identification of Langerhans cells in mouse epidermal cell suspension by scanning electron microscopy

The distribution of Ia^k antigens on suspended epidermal cells prepared from C3H/He mice by trypsinizing their ear skin was examined by the scanning immunoelectron microscopic method using antibody against the antigen and antibody bacteriophage T4 conjugates as a visual marker. Ia^k antigens were found to be distributed diffusely over a cell type with a relatively flat shape and a number of microvilli and ruffles. These cells are considered to be Langerhans cells. Significance of these findings is discussed.     

Origin of Langerhans cells in normal skin and chronic GVHD after hematopoietic stem‐cell transplantation

Chronic graft‐versus‐host disease (cGVHD) is a common complication following allogeneic stem‐cell transplantation (SCT). Past studies have implicated the persistence of host antigen‐presenting cells (APCs) in GVHD. Our objective was to determine the frequency of host Langerhans cells (LCs) in normal skin post‐SCT and ask if their persistence could predict cGVHD. Biopsies of normal skin from 124 sex‐mismatched T‐cell‐replete allogenic SCT recipients were taken 100 days post‐transplant. Patients with acute GVHD and those with <9 months of follow‐up were excluded and prospective follow‐up information was collected from remaining 22 patients. CD1a staining and X and Y chromosome in‐situ hybridization were performed to label LCs and to identify their host or donor origin. At 3 months, 59 ± 5% of LCs were host derived. The density of LCs and the proportion of host‐derived LCs were similar between patients that did or did not develop cGVHD. Most LCs in the skin remained of host origin 3 months after SCT regardless of cGVHD status. This finding is in line with the redundant role of LCs in acute GVHD initiation uncovered in recent experimental models.     

PUVA therapy decreases HLA-DR+ CD1a+ Langerhans cells and epidermal cell antigen-presenting capacity in human skin, but flow cytometrically-sorted residual HLA-DR+ CD1a+Langerhans cells exhibit normal alloantigen-presenting function

We have investigated the effects of PUVA therapy on human Langerhans cell (LC) immunophenotype and function. Epidermal sheets were obtained from exposed, and control shielded, forearm skin at the end of a course of PUVA therapy, in patients receiving treatment routinely for a variety of dermatoses. PUVA therapy decreased the overall number of HLA-DR+CDIa+ LCs in epidermal sheets, and in epidermal cell (EC) suspensions examined using a fluorescence activated cell sorter (FACS). PUVA therapy also reduced the overall EC allostimulatory capacity in the allogeneic epidermal cell-lymphocyte reaction (ELR), and the capacity of ECs to present tetanus toxoid to, and augment concanavalin A-mediated stimulation of, lymphocytes in the autologous ELR. Depressed allostimulation by ECs from PUVA-treated skin could not be restored by indomethacin (added to block prostaglandin synthesis). The reductions in LC numbers and EC allostimulatory capacity varied according to dose, and time since cessation, of PUVA therapy, and in individual patients were of comparable degree. By contrast, the allostimulatory capacity of residual LCs from PUVA-treated skin (purified using the FACS) did not differ from that of purified control LCs. PUVA-induced suppression of cutaneous immune responses, therefore, results at least in part from an overall impairment of EC antigen-presenting capacity. Residual HLA-DR+CDIa+ LCs in PUVA-treated skin which retain their alloantigen-presenting function may represent a subgroup of PUVA-resistant LCs; alternatively, these cells may be as yet unaffected because they have only recently migrated into the epidermis.     

联用密度梯度离心与免疫磁珠法分离纯化人表皮朗格汉斯细胞

目的:探讨获取较高纯度和活性的人表皮朗格汉斯细胞(LC)的实验方法。方法:联合采用密度梯度离心和免疫磁珠的方法分离纯化人表皮LC,将分选后的细胞用结合有异硫氰酸荧光素(FITC)的鼠抗人CD1a单抗标记,经流式细胞仪检测LC的纯化率,采用0.4%锥虫蓝染色检测细胞的活性。结果:分选所得LC纯化率为84.48%,锥虫蓝染色显示细胞活性>95%。结论:联用密度梯度离心和免疫磁珠法,可获得较高纯度、有活性的LC细胞,且具有操作简单、易于无菌条件下分离纯化的优点。     

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     

Control of the differentiation state and function of human epidermal Langerhans cells by cytokines in vitro

Langerhans cells can originate in vitro from immature precursors stimulated with granulocyte macrophage-colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF)-alpha and stem cell factor (SCF). We asked whether these cytokines also control the differentiation state of Langerhans cells within the epidermis and upon leaving this tissue. We harvested sheets of human epidermis by controlled dispase hydrolysis of keratomes, cultured them in RPMI and 10% fetal calf serum for 48 h and analysed the sheets and the cells migrated spontaneously into the medium, most of which were Langerhans cells containing Birbeck granules. By flow cytometry, the intensity of CD1a expression was reduced quite evenly among Langerhans cells migrated from sheets within 48 h. The cells in the sheets underwent loss of dendrites, with a significant reduction in the cell perimeter that was prevented by GM-CSF and TNF-alpha together. Either of these cytokines induced expression of CD18 by cells in the sheets and those in the medium. Moreover, TNF-alpha induced expression of CD54 by cells in the medium, but not by those retained in the sheets, whereas human SCF induced, dose dependently, expression of CD54 by cells in the sheets, but not from those in the medium. The proliferation of allogeneic lymphocytes was much higher when stimulating Langerhans cells were harvested from cultures with any cytokine, rather than from cultures without cytokines. We conclude the following: (i) GM-CSF and TNF-alpha help to maintain full differentiation of Langerhans cells within the epidermis; (ii) cytokine influence on Langerhans cells adhesiveness is in part context dependent; and (iii) pretreatment with cytokines influences positively the number or accessory activity of Langerhans cells on lymphocytes during subsequent mixed leucocyte reaction.     

Roles of CD4+ and CD8+ cells in UVB-induced suppression of splenic alloantigen presenting function

Abstract Whole body ultraviolet light (UV) radiation causes systemic immunosuppression. Splenic antigen-presenting cell (APC) activity is decreased by UV radiation. To determine whether splenic CD4⁺ or CD8⁺ cells are involved in the UV-induced depression of splenic alloantigen presenting function, we investigated the effect of in vivo UV radiation on the splenic stimulatory function in allogeneic mixed lymphocyte reaction in mice after the elimination of CD4⁺ or CD8⁺ cells by administering anti-CD8 or anti-CD4 Ab. Ab-treated and non-treated mice were exposed to UVB radiation (2.5 k.J/m²) twice or eight times. Two exposures to UVB radiation significantly suppressed the splenic alloantigen-presenting function of mice previously treated with anti-CD4 Ab, but hardly affected that of anti-CD8 Ab-treated or non-treated mice 2 days after the last radiation. On the other hand, eight exposures to UVB radiation suppressed this function in all mice. FACS analysis revealed that the UV induced suppression is not associated with a significant decrease in the number of IA⁺ cell, as stimulator cells. It is suggested that CD4⁺ cells are somewhat preventive of the UV-induced depression of splenic alloantigen-presenting function.     

Concurrent cutaneous localization of Langerhans cell sarcoma and chronic lymphocytic leukemia/small lymphocytic lymphoma in a patient with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma

The phenomenon of histiocytic/dendritic cell sarcomas arising through transformation of a pre-existed lymphoproliferative disease is called transdifferentiation. Langerhans cell sarcoma transdifferentiating from chronic lymphocytic leukemia/small lymphocytic lymphoma is extremely rare and all the reported cases were localized in lymph nodes. We present a case of concurrent cutaneous localization of Langerhans cell sarcoma and chronic lymphocytic leukemia/small lymphocytic lymphoma, in which the chronic lymphocytic leukemia/small lymphocytic lymphoma preceded the development of the Langerhans cell sarcoma. A cutaneous lesion from a 63-year-old patient with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma was biopsied. The histologic examination revealed a mixture of two cell populations infiltrating diffusely the dermis. The first was composed of small lymphoid cells with somewhat monotonous appearance and mild nuclear atypia positive for PAX5, CD79a, CD20, CD23, CD5, and LEF1. The second was composed of large cells with abundant cytoplasm and pleomorphic nuclei. These cells were positive for CD1a, CD207, and S100 protein and exhibited a high mitotic rate and a high MIB-1 immunostaining index. Therefore, two different entities, chronic lymphocytic leukemia/small lymphocytic lymphoma and Langerhans cell sarcoma, were detected in the same skin fragment. The patient died 3 years after initial diagnosis of chronic lymphocytic leukemia/small lymphocytic lymphoma.     

Detection of Epstein-Barr virus in cutaneous and lymph nodal anaplastic large cell lymphomas (Ki-l+)

Summary Epstein-Barr virus (EBV) is a gamma DNA herpes virus which is thought to play a part in the pathogenesis of some non-Hodgkin's lymphomas in individuals with or without immunodeficiency. We investigated 16 lymph nodal and 12 cutaneous anaplastic large cell lymphomas (ALCLs) (Ki-1 ⁺), all of which were in patients without immunodeficiency, for the presence of EBV genomes. The highly sensitive polymerase chain reaction (PCR) technique was employed for detection of viral DNA in extracts from formalin-fixed, paraffin-embedded tissue sections. In addition, we performed radioactive and non-radioactive in situ hybridization (ISH) for localization of EBV at the single cell level. EBV-DNA was demonstrated by PCR in five cases of nodal ALCLs (31 %). All cutaneous ALCLs were negative. EBV-encoded small nuclear RNAs (EBERs) could be identified by ISH in the tumour cells of one of the five EBV-DNA-positive patients. Our results further support the concept that EBV may be involved in the development of a proportion of nodal ALCLs, hut not in cutaneous ALCLs.     

Modulation of phospholipase A2 activity in extracts of lesion-free psoriatic epidermis by alkaline phosphatase and a protein phosphatase inhibitor

Phospholipase A₂ activity is raised in non-lesional psoriatic epidermis compared with normal epidermis. It has been shown that the activity of this enzyme is controlled by an inhibitory protein the inhibitory effect of which is increased by dephosphorylation. Treatment of epidermal extracts with alkaline phosphatase reduced the phospholipase A₂ activity, both in normal and in lesion-free psoriatic epidermis. Inclusion of pyrophosphate, a protein phosphatase inhibitor, in the homogenizing medium caused the activity of phospholipase A₂ in epidermal extracts from normal and lesion-free epidermis to be raised to the same high level. These results are consistent with the hypothesis that the raised phospholipase A₂ activity in psoriatic epidermis is due to hyperphosphorylation of an endogenous inhibitor as a result of defective control of a phosphorylation/dephosphorylation mechanism. The relevance of these findings to other work is discussed.     

Deregulation of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in basal cell carcinoma

Background  Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population.
Objectives  To determine the involvement of the tumour suppressor genes p14^ARF, p15^INK4b, p16^INK4a and p53 in BCC.
Methods  We investigated the integrity of the CDKN2A locus in 15 BCC samples by analysing the presence of allelic imbalance/loss of heterozygosity (LOH). Moreover, we studied the mRNA expression levels of the tumour suppressor genes p14^ARF, p15^INK4b, p16^INK4a and p53 in the BCC samples and compared them with mRNA levels in the corresponding normal tissue. The presence of mutations was examined by sequencing for exons 1a and 2 of p16^INK4a.
Results  We found LOH in one BCC sample for the marker D9S1748. A polymorphism (G442A) of exon 2 was detected in three cases. p14^ARF, p15^INK4b and p53 presented high expression levels, whereas p16^INK4a exhibited low mRNA levels compared with the corresponding normal tissue. Significant correlations were detected among the genes studied.
Conclusions  Our results demonstrate a different expression profile between p16^INK4a and p14^ARF, p15^INK4b and p53 in BCC. Moreover, we found a low percentage of LOH and of a polymorphic sequence variant (Ala148Thr) for the CDKN2A locus.     

A randomized, single-blind comparison of topical clindamycin + benzoyl peroxide (Duac®) and erythromycin + zinc acetate (Zineryt®) in the treatment of mild to moderate facial acne vulgaris

BACKGROUND: Antibiotics are often combined with other agents to provide topical acne treatments that are effective against both inflammatory and non-inflammatory lesions and minimize the development of antibiotic resistance. OBJECTIVES: To compare the clinical effectiveness of two combination treatments for facial acne: a ready mixed, once daily gel containing clindamycin phosphate (1%) plus benzoyl peroxide (5%) (CDP + BPO) and a twice daily solution of erythromycin (4%) plus zinc acetate (1.2%) (ERY + Zn). METHODS/PATIENTS: In this assessor-blind, randomized study, 73 patients were treated with CDP + BPO once daily and 75 patients with ERY + Zn twice daily. The treatment period was 12 weeks and lesion counts and global improvement were assessed at weeks 1, 2, 4, 8 and 12. RESULTS: CDP + BPO showed an earlier onset of action with a faster significant reduction in total lesion counts than ERY + Zn. The proportion of patients with at least a 30% improvement in non-inflammatory lesions at week 1 was 31.5% for CDP + BPO and 17.3% for ERY + Zn; the corresponding percentages for inflammatory lesions were 39.7% and 29.3%. A difference was also observed at week 2 (53.4% vs. 36.0% for non-inflammatory lesions and 72.6% vs. 53.3% for inflammatory lesions). The trend in favour of CDP + BPO, although less marked, continued to the end of the study, with reductions in the total lesion count at endpoint of 69.8% for CDP + BPO group and 64.5% for ERY + Zn group. Both treatments were well tolerated. CONCLUSIONS: CDP + BPO and ERY + Zn are effective treatments for acne but CDP + BPO has an earlier onset of action that should improve patient compliance.     

Low to high Ca2+-switch causes phosphorylation and association of desmocollin 3 with plakoglobin and desmoglein 3 in cultured keratinocytes

Abstract:  Although desmocollins (Dscs) and desmogleins (Dsgs) are known to be bound to each other to form desmosomes, neither their interactions nor regulations that occur in human keratinocytes grown in low and high Ca²⁺medium has been determined. In this study, we investigated whether Dsc3 interacts with Dsg3 in a cell line of human squamous cell carcinoma keratinocytes (DJM-1) grown in low (0.05 m m ) or high (1.27 m m ) Ca²⁺medium. Anti-Dsc3 monoclonal antibody did not co-immunoprecipitate Dsg3 nor plakoglobin with Dsc3 in low Ca²⁺culture, whereas it co-immunoprecipitated plakoglobin already at 10 min and Dsg3 at 60 min after Ca²⁺-switch in association with Dsc3 phosphorylation at serine residues. These results suggest that both the binding of Dsc3 to plakoglobin and Dsc3 phosphorylation are involved in Dsc3 binding to Dsg3 during Ca²⁺-induced desmosome assembly.     

Expression of dipeptidyl-peptidase IV (CD26) on CD8+ T cells is significantly decreased in patients with psoriasis vulgaris and atopic dermatitis

T cells play a major role in inflammatory skin disorders such as psoriasis vulgaris and atopic dermatitis. They are both active on the level of cell-to-cell interaction and by the secretion of pro-inflammatory mediators. CD26 is a lymphocyte membrane-associated dipeptidyl peptidase IV (DPP IV), which is able to inactivate chemokines such as RANTES or eotaxin by cleaving dipeptides from the NH2-terminus of proteins. We investigated the expression of CD26 on CD4+ and CD8+ peripheral blood T cells in patients with psoriasis and atopic dermatitis. In addition PASI and SCORAD as a measure of disease severity were determined in each patient at the time of blood drawing. Thirty patients with psoriasis, 15 with atopic dermatitis and 17 age- and sex-matched healthy persons were investigated by two-colour flow cytometry using epitope-specific monoclonal antibodies. Our results revealed, that there is a significant decrease (P<0.05) of CD26 expression on CD8+ T cells in both psoriasis (7.7%+/-3.3, mean and SD, n=30) and atopic dermatitis patients (7.9%+/-3.7, mean and SD, n=15) compared to the control population (11.58%+/-5.0, mean and SD, n=17). However, there was no correlation to disease severity as determined by PASI and SCORAD, respectively. Since CD26 can be regarded as an anti-inflammatory principle the decreased expression in psoriasis and atopic dermatitis patients may lead to a dysbalance in favour of pro-inflammatory mediators in both clinical conditions.