Heinz-Joachim Radzun

EXPRESSION OF METALLOPROTEINASE 2 AND 9 AND THEIR INHIBITORS IN RENAL CELL CARCINOMA

Degradation of the extracellular matrix is necessary for invasion and metastasis by cancer cells. Two gelatinolytic matrix metalloproteinase enzymes, MMP-2 and MMP-9, are supposed to be key enzymes in this process. The purpose of this study was to correlate the presence of MMP-2, MMP-9 and their inhibitors with the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 RNA using reverse transcriptase PCR technique with tumor stage in 17 samples of renal cell carcinoma. The ratio of tissues expressing MMP-2 and MMP-9 to those expressing TIMP-1 and TIMP-2 was defined to be 1 in normal kidney tissue. This MMP:TIMP ratio was significantly increased to 2.43 (standard deviation, SD = 0.8) in locally confined renal cell carcinoma and to 4.86 (SD = 1.1) in advanced carcinoma (p <0.01). In primary tumor cell lines the ratio of MMP:TIMP expression was 3.44 (SD = 0.6). These data suggest that the balance of MMP-2 and MMP-9 to TIMP-1 and TIMP-2 expression is an essential factor in the aggressiveness of renal cell carcinoma.

Expression of IFNγ, coexpression of TNFα and matrix metalloproteinases and apoptosis of T lymphocytes and macrophages in granuloma annulare

Abstract Granuloma annulare, a prototype noninfectious granulomatous dermatitis, is morphologically characterized by a necrobiotic core surrounded by a cellular infiltrate. Because of many morphological similarities to tuberculosis, granuloma annulare has been suggested to represent a delayed-type hypersensitivity (Th1) reaction in the course of which inflammatory cells elicit matrix degradation. In the present study we (1) investigated the expression of interferon-Á as the most important Th1-associated cytokine, (2) sought in situ evidence for the coexpression of the proinflammatory cytokine tumor necrosis factor-· and cytokine-regulated matrix metalloproteinases 2 (gelatinase A) and 9 (gelatinase B), and (3) sought to determine whether shrunken cells seen within necrobiotic areas of granuloma annulare are apoptotic cells. In situ hybridization combined with immunofluorescence showed that large numbers of infiltrating CD3 + lymphocytes express interferon-Á. Application of catalyzed signal amplification in immunodetection revealed that the vast majority of CD3 + lymphocytes and CD68 + macrophages contained tumor necrosis factor-·. Immunohistochemistry demonstrated that macrophages producing tumor necrosis factor-· coexpress matrix metalloproteinases 2 and 9. In situ end-labeling combined with immunofluorescence detected few apoptotic T cells in perivascular regions and numerous apoptotic macrophages within necrobiotic areas. These results suggest that in granuloma annulare interferon-Á + Th-1 lymphocytes may cause a delayed-type hypersensitivity reaction whereby macrophages are differentiated to aggressive effector cells expressing tumor necrosis factor-α and matrix metalloproteinases. In parallel, activation-induced apoptosis in lymphocytes and macrophages may serve to restrict the destructive potential of the inflammatory cells.

Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas

Rapidly growing tumors often develop necrosis. In the present study the expression of vascular endothelial growth factor (VEGF) was investigated and compared to microvessel density and necrosis of renal cell carcinomas. In the tumor-host interface the microvessel density was significantly increased compared to central tumor areas. Tumor necrosis was associated with a decrease of microvessel density and an increase of the VEGF protein expression within the perinecrotic rim. VEGF protein was focally upregulated in vital tumor tissue. An increase of the apoptotic rate of endothelia and vital tumor tissue in tumors with necrosis could not be detected. VEGF(121,165) mRNA was decreased in proliferatively active carcinomas compared to less proliferative tumors. Multicellular renal cell cancer spheroids as a model of chronic hypoxia developed central apoptosis but no necrosis. VEGF was upregulated in the spheroid. Tumor microvessels expressed matrix metalloproteinase -2 and -9 and an incomplete pericyte covering in comparison to tumor-free tissue indicating immature active angiogenesis. We conclude that highly proliferative renal cell carcinomas outgrow their vascular supply and develop chronic hypoxia inducing a decrease of proliferation and an increase of VEGF expression. However, chronic hypoxia does not cause significant necrosis or apoptosis. Tumor necrosis is more likely induced by acute hypoxia due to immature microvessels. Furthermore, VEGF expression associated with concomitant tumor necrosis may help identify renal cell carcinomas susceptible to antiangiogenic therapy.

T lymphocytes and altered keratinocytes express interferon-γ and interleukin 6 in lichen planus

Abstract Lichen planus is asumed to represent a delayed hypersensitivity reaction, in the course of which cytokines control the proliferation and differentiation of cytotoxic T lymphocytes which attack the epidermis and cause apoptosis of undifferentiated keratinocytes. Since interferon-γ and interleukin 6 are known to be markedly generated in lichen planus, we investigated the cellular localization of these cytokines in affected skin/oral mucosa biopsy specimens using in situ hybridization for interferon-γ and in situ reverse transcription-polymerase chain reaction for interleukin 6 mRNA. In the upper subepithelial connective tissue interferon-γ mRNA was noted within proliferating CD3 + T lymphocytes. In this tissue compartment interleukin 6 mRNA was detected in infiltrating CD4 + and CD8 + T lymphocytes. In the epithelium, expression of interferon-γ mRNA and interleukin 6 mRNA was observed in the basal and suprabasal keratinocytes of altered skin/oral mucosa. In contrast, normal skin did not reveal any interferon-γ or interleukin 6 expression, although a few CD4 + and CD8 + T lymphocytes were noted in the dermis as well as the epidermis. These findings indicate that in lichen planus the proinflammatory cytokines interferon-γ and interleukin 6 are produced not only by activated T lymphocytes but also by altered keratinocytes, and suggest that stimulated keratinocytes may amplify the course of lichen planus.

Expression of the T-cell chemoattractant chemokine lymphotactin in Crohn's disease.

Recruitment of lymphocytes is a prominent feature of the inflammatory process in Crohns disease (CD). The present study was undertaken to investigate the expression of the novel lymphocyte-specific chemoattractant lymphotactin (Lptn) as a potential regulatory factor for the recruitment of T cells in CD. The expression of Lptn mRNA was quantified in resection specimens of patients with CD in comparison to normal controls without signs of inflammation by real-time quantitative reverse transcriptase-polymerase chain reaction and localized by nonradioactive in situ hybridization. Furthermore, the phenotype of cells expressing Lptn mRNA was characterized. In contrast to normal controls Lptn mRNA was significantly increased in tissue samples affected by CD. Cells expressing Lptn were identified as T cells, mast cells, and unexpectedly dendritic cells. Lptn mRNA was found to be up-regulated on stimulation with phorbol-12-myristate-13-acetate and concanavalin A in T cells isolated from peripheral blood, which could be prevented by dexamethasone, cyclosporine A, and FK506. A similar regulation mechanism could be identified for the Lptn receptor GPR-5 in peripheral T cells. In addition, Lptn mRNA expression could be induced in mature monocyte-derived dendritic cells. The results indicate that local expression of Lptn by activated T cells and to a lesser extent by mast cells and dendritic cells represents a key regulator for lymphocyte trafficking and maintenance of the inflammatory process observed in CD, which might be partly mediated through an autocrine/paracrine pathway of activated T cells.

Expression of acute and late-stage inflammatory antigens, c-fms, CSF-1, and human monocytic serine esterase 1, in tumor-associated macrophages of renal cell carcinomas

Purpose: Tumor cells influence the differentiation of infiltrating macrophages. In the present study, the differentiation of macrophages in renal cell carcinomas was investigated with special regard to their possible role in tumor growth and spread. Methods: Macrophages were characterized by means of immunohistochemistry of the Ki-M1P, 25F9, MRP8, MRP14, and MRP8/14 antigens and by means of in situ hybridization of CSF-1, its c-fms-coded corresponding receptor, and human monocytic serine esterase-1 (HMSE-1) mRNA. Macrophage subgroups were quantified within central tumor tissue, the corresponding tumor host interface, and tumor-free tissue and correlated with tumor necrosis, fibrosis, and tumor stage and grade. Results: Macrophage density was much higher within tumor tissue and the tumor/host interface than in tumor-free tissue. Well-differentiated carcinomas showed a lower degree of macrophage density than less-differentiated carcinomas. Tumor-associated macrophages could be divided into an active inflammatory type (MRP14+, MRP8/14+) and into a late-phase inflammatory type (25F9+, MRP8+). Necrosis was seen in less-differentiated carcinomas and associated with a significantly increased density of MRP14+ macrophages, which, however, did not correlate with the extent of necrosis. The density of 25F9+ macrophages was correlated with an extensive connective tissue formation and an advanced tumor stage. c-fms, CSF-1, and HMSE-1 mRNA expression did not discriminate between the macrophage subgroups. Conclusions: Tumor-associated macrophages of the late-stage inflammatory type potentially support the spread of renal cell cancer. CSF-1 derived from tumor cells and macrophages acts as a monocyte attractant and induces macrophage differentiation able to modulate the extracellular matrix rather than to exert cytotoxicity.

Chemokine-mediated distribution of dendritic cell subsets in renal cell carcinoma

BackgroundRenal cell carcinoma (RCC) represents one of the most immunoresponsive cancers. Antigen-specific vaccination with dendritic cells (DCs) in patients with metastatic RCC has been shown to induce cytotoxic T-cell responses associated with objective clinical responses. Thus, clinical trials utilizing DCs for immunotherapy of advanced RCCs appear to be promising; however, detailed analyses concerning the distribution and function of DC subsets in RCCs are lacking.MethodsWe characterized the distribution of the different immature and mature myeloid DC subsets in RCC tumour tissue and the corresponding normal kidney tissues. In further analyses, the expression of various chemokines and chemokine receptors controlling the migration of DC subsets was investigated.ResultsThe highest numbers of immature CD1a+ DCs were found within RCC tumour tissue. In contrast, the accumulation of mature CD83+/DC-LAMP+ DCs were restricted to the invasive margin of the RCCs. The mature DCs formed clusters with proliferating T-cells. Furthermore, a close association was observed between MIP-3α-producing tumour cells and immature CCR6+ DC recruitment to the tumour bed. Conversely, MIP-3β and SLC expression was only detected at the tumour border, where CCR7-expressing T-cells and mature DCs formed clusters.ConclusionIncreased numbers of immature DCs were observed within the tumour tissue of RCCs, whereas mature DCs were found in increased numbers at the tumour margin. Our results strongly implicate that the distribution of DC subsets is controlled by local lymphoid chemokine expression. Thus, increased expression of MIP-3α favours recruitment of immature DCs to the tumour bed, whereas de novo local expression of SLC and MIP-3β induces accumulation of mature DCs at the tumour margin forming clusters with proliferating T-cells reflecting a local anti-tumour immune response.

Expression and function of protein phosphatase PP2A in malignant testicular germ cell tumours

Testicular germ cell tumours (TGCT) represent the most common malignancy in young males. We reported previously that two prototype members of the mitogen‐activated protein kinase (MAPK) family, the MAPK ERK kinase (MEK) and extracellular signal‐regulated kinase (ERK), are inactive in malignant testicular germ cells and become active after drug stimulation, leading to apoptosis of tumour cells. In this study, we asked whether the protein phosphatase PP2A, a known inhibitor of the MEK–ERK pathway, participates in the proliferation and/or apoptosis of primary TGCT (n = 48) as well as two TGCT cell lines (NTERA and NCCIT). Quantitative RT–PCR, immunohistochemistry, western blot analyses and phosphatase assay indicate that primary TGCT as well as TGCT cell lines express PP2A and that PP2A is active in TGCT cell lines. The inhibition of PP2A by application of two PP2A inhibitors, cantharidic acid (CA) and okadaic acid (OA), results in a significant increase in caspase‐3‐mediated apoptosis of TGCT cell lines. Thereby, PP2A inhibition was accompanied by phosphorylation and activation of MEK and ERK. Functional assays using the MEK inhibitor PD98059 demonstrated that the phosphorylation of MEK and ERK was required for the induction of caspase‐3‐mediated apoptosis of malignant germ cells. Thus, our data suggest that inhibition of PP2A mediates its apoptosis‐inducing effect on TGCT through activation of the MEK–ERK signalling pathway that leads to caspase‐3‐mediated apoptosis of tumour cells. In addition our results support previous observations that PP2A exerts an anti‐apoptotic effect on malignant tumour cells. Copyright

Cell renewal, cell differentiation and programmed cell death (apoptosis) in pilomatrixoma.

Summary Pilomatrixoma is a benign tumour of the cutaneous adnexa. Histologically. pilomatrixoma comprises masses of immature basophilic cells, small numbers of polygonal squamoid cells, few transitional cells, and clusters of ‘shadow cells’. The mechanism leading to the formation of shadow cells is still unknown. Skin biopsy specimens of pilomatrixoma (n= 15) were studied histologically, immuno‐hislologically, and by applying the in situ end‐labelling technique. The basal layer of the basophilic cells included most of the proliferating cells with high expression of bcl‐2 and cytokeratin 19. The overlying basophilic cells showed a negligible mitotic activity, a high significant accumulation of p53 protein, and a heterogeneous, but progressive loss of bcl‐2 and cytokeratin 19. They developed either into squamoid cells or into transitional cells. The squamoid cells were characterized as differentiated cells resembling mature keratinocytes of stratified mucosa. The transitional cells could be shown to represent apoptotic cells proceeding to shadow cells. The data suggest that apoptosis is the main mechanism leading to the development of the dead shadow cells and is most probably responsible for the banal biological behaviour of pilomatrixoma. Apart from that, pilomatrixoma represents a suitable biological model to study apoptosis in humans.

Expression of Lymphotoxin-α by Keratinocytes: A Further Mediator for the Lichenoid Reaction

Objective: Lichen planus (LP) represents a disease in which autoimmune mechanisms mediated by Th1 T cells are involved. Lymphotoxin-α (LT-α) represents a Th1 cytokine with proinflammatory activities in LP, as has recently been demonstrated for interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Methods: Expression of LT-α mRNA was investigated by RT-PCR and nonradioactive in situ hybridization. Double staining methods were applied to characterize the phenotype of cells expressing LT-α. Cell stimulation experiments were performed on the transformed squamous cell line HaCaT. Results: In contrast to normal skin, LT-α-specific RT-PCR products were found in all cases of LP. Cells in the inflammatory infiltrate expressing LT-α were identified as mainly T cells and mast cells, as shown by in situ hybridization. Furthermore, predominant LT-α mRNA expression could be observed in lesional keratinocytes adjacent to the band-like inflammatory infiltrate. In cell stimulation experiments, it could be shown that IFN-γ induces LT-α and TNF-α mRNA in the human squamous cell line HaCaT, concomitant with upregulation of MHC class II and intercellular adhesion molecule-1, which could also be observed on lesional keratinocytes in LP. Conclusions: In LP, LT-α mRNA is predominantly expressed by lesional keratinocytes and to a lesser extent by inflammatory cells. Induction of LT-α in keratinocytes is closely related to the expression of TNF-α and MHC class II. The loci of TNF-α and LT-α map to MHC class III on chromosome 6, which is closely linked to the MHC class II gene locus. Our results suggest that stimulation of keratinocytes with IFN-γ results in the upregulation of proinflammatory cytokines such as LT-α and TNF-α as well as MHC class II, which map to the same gene region of immunoregulatory genes on chromosome 6 and may be involved in the induction and maintenance of the disease.