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The cyclin-dependent kinase inhibitor 2A (CDKN2A) gene on chromosome 9p21 encodes p16 (INK4A), the inhibitor of the CDK4/retinoblastoma (Rb) cell proliferation pathway, as well as p14 (ARF), which controls p53-dependent pathways. Inactivation of p16 has previously been associated with the prognostically unfavourable primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT). In this work, we analysed 22 tumors [nine primary cutaneous follicle centre lymphomas (PCFCL), seven primary cutaneous marginal zone lymphomas (PCMZL) and six PCLBCL, LT] not only for alterations of the p16 gene but also for p14, p53 and Rb by fluorescence in situ hybridization (FISH) and immunohistochemistry. In most PCLBCL, LT (4/6) alterations of CDKN2A (two biallelic deletions, one monoallelic deletion and one trisomy 9) and in addition the highest frequency of deletions of p53 (3/6) and Rb (3/6) were detected. p16 was not expressed but very high levels of phosphorylated Rb, indicating a functional effect of genomic CDKN2A alterations on the protein level in PCLBCL, LT. Regarding the p14/p53 axis, PCLBCL, LT showed a variable expression. Neither PCFCL nor PCMZL showed alterations of CDKN2A and also deletions of p53 or Rb were extremely rare in these subtypes. Exclusively in PCMZL, p53 protein was consistently lacking. In conclusion, only PCLBCL, LT is characterized by a high frequency of aberrations of the CDKN2A network components in both important tumor suppressor pathways regulated by the CDKN2A gene. Moreover, PCLBCL, LT appears to be distinguishable from PCMZL not only by its level of p53 expression but also by its stage of Rb phosphorylation. The latter may also apply to a subgroup of PCFCL.

CXCL8 plays a major role in cell recruitment to sites of inflammation. Apart from neutrophils, little is known, however, about the cellular distribution and regulation of CXCL8 receptors in cells involved in acquired and adaptive immune responses. In previous studies, we have demonstrated the extracellular expression and function of CXCR1/2 on mast cells and also detected an intracellular pool of CXCR1/2. Here, we have addressed the question of receptor regulation during stimulation of human mast cells (HMC-1 cell line) and have studied T cells in comparison. Cell permeabilization was performed to detect both surface and possible intracellular receptor pools. HMC-1 cells stained positive for both receptors on the cell surface (CXCR1, 50%; CXCR2, 51%) and also after cell permeabilization (CXCR1, 86%; CXCR2, 74%). Similarly, T cells exhibited both cell-surface receptor expression (CXCR1, 30%; CXCR2, 23%) and higher total receptor expression (CXCR1, 50%; CXCR2, 36%), although overall values were lower than that in HMC-1 cells. On immunoblot, molecular weights of extra- and intracellular receptors on mast cells were the same, excluding altered receptor glycosylation. On stimulation with phorbol 12-myristate 13-acetate plus calcium ionophore, a time-dependent decrease of surface-membrane receptors was observed in both cell types, while total receptor remained the same, suggesting that receptor shedding is not involved. The kinetics of membrane receptor internalization and replenishment differed for the two cell types. Furthermore, receptor internalization was associated with decreased F-actin polymerization, a basic prerequisite for cell migration. These findings demonstrate for the first time the expression of extra- and intracellular CXCR1/2 receptors on T cells and delineate the dynamics of CXCR1/2 receptors on mast cells and T cells. Furthermore, they suggest a cell-type-specific and finely tuned regulation of chemokine responses at the receptor level in the context of inflammation.

Psoriasis is a chronic inflammatory skin disorder characterized by accumulation of Th1-type T cells and neutrophils, regenerative keratinocyte proliferation and differentiation, and enhanced epidermal production of antimicrobial peptides. The underlying cause is unknown, but there are some similarities with the immunologic defense program against bacteria. Development of psoriasiform skin lesions has been reported after administration of granulocyte colony-stimulating factor (G-CSF), a cytokine induced in monocytes by bacterial antigens. To further investigate the relation between this type of cytokine-induced dermatitis and psoriasis, we analyzed the cutaneous cytokine profile [tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, transforming growth factor-beta1 (TGF-beta1), interleukin-10 (IL-10), IL-12p35 and p40, and IL-8] and expression of markers of epidermal activation [Ki-67, cytokeratin-16, major histocompatibility complex (MHC) class II, intercellular adhesion molecule-1 (ICAM-1)] in a patient who developed G-CSF-induced psoriasiform dermatitis by using quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistology. The histologic picture resembled psoriasis with regard to epidermal hyperparakeratosis and the accumulation of lymphocytes in the upper corium. CD8(+) T cells were found to infiltrate the epidermis which was associated with an aberrant expression of Ki-67, cytokeratin-16, MHC class II, and ICAM-1 on adjacent keratinocytes. As compared to normal skin (n = 7), there was an increased expression of TNF-alpha, IL-12p40, and IL-8, a decreased expression of TGF-beta1, and a lack of IL-10, similar to the findings in active psoriasis (n = 8). Therefore, G-CSF may cause a lymphocytic dermatitis that, similar to psoriasis, is characterized by a pro-inflammatory Th1-type cytokine milieu and an epidermal phenotype indicative of aberrant maturation and acquisition of non-professional immune functions.

In atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cytokines interleukin-4 (IL-4) and IL-5 but not interferon-gamma (INF-gamma). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both Th1 and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteronyssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (p < 0.05). The difference was more significant with specific TCC than with non-specific TCC. Furthermore, there was a positive correlation between the production of IL-10 and that of IL-4 and IL-5, respectively. In addition, the amount of IL-4 and IL-5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood-derived TCC IL-10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2-like secretion pattern not only with respect to IL-4 and IL-5 but also regarding IL-10.

The cyclin-dependent kinase inhibitor 2A (CDKN2A) gene on chromosome 9p21 encodes p16 (INK4A), the inhibitor of the CDK4/retinoblastoma (Rb) cell proliferation pathway, as well as p14 (ARF), which controls p53-dependent pathways. Inactivation of p16 has previously been associated with the prognostically unfavourable primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT). In this work, we analysed 22 tumors [nine primary cutaneous follicle centre lymphomas (PCFCL), seven primary cutaneous marginal zone lymphomas (PCMZL) and six PCLBCL, LT] not only for alterations of the p16 gene but also for p14, p53 and Rb by fluorescence in situ hybridization (FISH) and immunohistochemistry. In most PCLBCL, LT (4/6) alterations of CDKN2A (two biallelic deletions, one monoallelic deletion and one trisomy 9) and in addition the highest frequency of deletions of p53 (3/6) and Rb (3/6) were detected. p16 was not expressed but very high levels of phosphorylated Rb, indicating a functional effect of genomic CDKN2A alterations on the protein level in PCLBCL, LT. Regarding the p14/p53 axis, PCLBCL, LT showed a variable expression. Neither PCFCL nor PCMZL showed alterations of CDKN2A and also deletions of p53 or Rb were extremely rare in these subtypes. Exclusively in PCMZL, p53 protein was consistently lacking. In conclusion, only PCLBCL, LT is characterized by a high frequency of aberrations of the CDKN2A network components in both important tumor suppressor pathways regulated by the CDKN2A gene. Moreover, PCLBCL, LT appears to be distinguishable from PCMZL not only by its level of p53 expression but also by its stage of Rb phosphorylation. The latter may also apply to a subgroup of PCFCL.

Please cite this paper as: Enhanced T-cell activation by immature dendritic cells loaded with HSP70-expressing heat-killed melanoma cells. Experimental Dermatology 2010; 19: 108–116. Abstract: Vaccination protocols that utilize dendritic cells (DCs) to elicit therapeutic immunity against tumors are the subject of intense research. Given that the capacity of DCs to cross-present antigens is physiologically low, there is considerable interest to develop strategies that enhance that pathway. In order to best exploit the enhanced cross-presentation of antigens bound to heat shock protein 70 (HSP70), we analysed melanoma cell preparations for their HSP70 expression. Western blotting revealed strong upregulation of HSP70 after heat-killing in contrast to UV-B irradiation. When the uptake of heat-killed necrotic cells by DCs at various levels of maturation was assessed, 61 ± 7% of immature DCs (iDCs) internalized fluorescence-labelled necrotic material. Apoptotic material from UV-B-irradiated cells was internalized by only 48 ± 5% of iDCs. Maturation-inducing cytokines did not affect the uptake when added simultaneously with the tumor cell preparations. Loading DCs with heat-necrotic or apoptotic melanoma cells slightly reduced CD83 expression while leaving CD208 (DC-LAMP) expression unchanged. As determined by IFN-γ-detecting enzyme-linked-immunospot assays, iDCs loaded with heat-killed melanoma cells activated autologous T cells most effectively when used without any further maturation, whereas DCs loaded with apoptotic material required maturation. In conclusion, HSP70-expressing melanoma cells could be generated by heat-killing. Loading iDCs with heat-killed melanoma cells resulted in a superior priming of autologous T cells in vitro.

Dendritic cells (DCs) have become popular candidates in cancer vaccination because of their crucial role in inducing T-cell responses. However, clinical studies greatly differ in their protocols for generating DCs and the efficacy in treating established tumors needs to be improved. We systematically analyzed DCs maturated by five different protocols for surface markers, the alloproliferative T-cell response, the DC survival after cytokine deprivation, the stability of surface markers under the influence of interleukin-10 (IL-10) and the DC cytokine secretion pattern. Monocyte-derived DCs were maturated by CD40-ligand (CD40-L), unmethylated cytosine-guanosine dinucleotides-oligodinucleotides (CpG-ODN), an inflammatory cytokine cocktail (ICC), a combination of ICC and CD40-L, or ICC, CD40-L and CpG-ODN. A high co-expression of DC maturation and costimulation markers was found after treatment with ICC plus CD40-L (69.3 +/- 9.6% CD83/CD80 double positive staining) and correlated with a significantly increased cell survival, a high expression of the antiapoptotic factor bcl-(XL), a stable CD83(high)/CD14(low) expression under the influence of IL-10, and a strong alloproliferative T-cell response. In conclusion, our data support the use of maturation protocols containing ICC plus CD40-L in order to generate highly mature, phenotypically stable, cell-death resistant, and T-cell stimulatory DCs for clinical application in cancer patients.

Decreased production of T helper type 1 (Th1) cytokines, such as interferon-gamma (IFN-gamma) or interleukin-2 (IL-2), is a hallmark of atopic diseases. While accessory signals from antigen-presenting cells may be missing, T cells themselves may be suppressed in their ability to produce substantial amounts of Th1 cytokines. We show, in this study, that T cell receptor (TCR)-activated T cells from atopic dermatitis (AD) patients proliferate less than control T cells and produce lower amounts of IFN-gamma and IL-2, but comparable amounts of IL-4. Because mice lacking the nuclear factor kappa B (NF-kappaB) transcription factors - p65 or c-Rel - show reduced Th1, but undisturbed Th2 responses, we investigated the role of c-Rel and p65 for Th1 cytokine production in T cells from healthy and severe AD patients. TCR-activated primary T cells from healthy donors treated with c-Rel antisense oligonucleotides produced lower levels of IL-2 and IFN-gamma and proliferated less efficiently than the corresponding control T cells. Moreover, transfection of primary T cells with c-Rel or p65 enhanced proliferation and production of IL-2 and IFN-gamma. Nuclear extracts of activated primary T cells from AD donors bound weakly to NF-kappaB-specific oligonucleotides, compared to extracts from healthy control T cells. Western blotting studies revealed that nuclear, but not cytosolic, extracts from T cells of AD patients lacked significant amounts of c-Rel and p65. T cell clones derived from AD patients failed to sufficiently translocate c-Rel and p65 into the nucleus following activation. Thus, impaired nuclear translocation of c-Rel and p65 may determine an impaired Th1 cytokine response in AD.

Background: Maturation of dendritic cells (DCs) influences important DC functions such as production of cytokines. Recently, DCs were identified as a source of interleukin-16 (IL-16), a chemotactic factor for DCs themselves, CD4+ T cells, and eosinophils. There is evidence that DC-derived IL-16 may contribute to the pathogenesis of atopic dermatitis (AD). Objective: To investigate the production of IL-16 during differentiation of monocytes into DCs in healthy individuals and patients with AD. Methods: IL-16 production was investigated by quantitative real-time RT-PCR, intracellular cytokine staining, immunoblotting, and ELISA. Results: DCs generated from peripheral monocytes by 5-day culture in the presence of IL-4 and granulocyte/macrophage colony-stimulating factor acquired the capability to synthesize, store, and secrete IL-16. Storage and release of IL-16 was further enhanced during final DC maturation induced by additional 3-day culture with tumor necrosis factor-α (TNF-α) and monocyte-conditioned medium. Maturation, as determined by up-regulation of CD83 and CD86 surface expression, and production of IL-16, but not production of IL-10 and IL-12p40 was impaired in day 8 DCs derived from AD patients compared to those from healthy donors. Stimulation of day 8 DCs from AD patients with TNF-α and IL-1β enhanced the expression of CD83 and CD86 and restored the production of IL-16. Conclusions: Signals involved in the activation and maturation of DCs enhance their capacity to produce IL-16. Functional abnormalities present in patients with AD at the monocyte level may account for impaired maturation and IL-16 production of monocyte-derived DCs.

In certain pathological conditions, such as atopic dermatitis, interleukin-4 (IL-4) can be detected in the skin. As the role of this cytokine in inflammatory skin lesions is not completely clear, we investigated its biological effects on skin keratinocytes. It was found that freshly isolated as well as cultured keratinocytes obtained from normal individuals express mRNA for the IL-4 receptor (IL-4R) and produce IL-4R protein, as determined by flow cytometry. Moreover, IL-4 induced a proliferative response in keratinocytes after 1 day of culture and enhanced B7/BB1 expression in these cells. B7-2 (CD86) mRNA and protein were neither detected on untreated nor IL-4 treated keratinocytes. In contrast to interferon-gamma (IFN-gamma), IL-4 did not induce ICAM-1 (CD54) or HLA-DR-expression. Keratinocytes which had been treated with IL-4 showed an enhanced ability to stimulate allogeneic T-cell proliferation in the presence of staphylococcus enterotoxin B (SEB), (p < 0.01). Neutralizing anti-B7/BB1 monoclonal antibodies did not block this effect. These results indicate that other molecules than B7/BB-1. HLA-DR or ICAM-1 on IL-4-activated keratinocytes may be involved in T-cell stimulation. In conclusion our results suggest that locally produced IL-4, besides modulating keratinocyte membrane molecules, may enable keratinocytes to interact with skin infiltrating lymphocytes.