Stefan Schweyer

The C5a receptor is expressed in normal renal proximal tubular but not in normal pulmonary or hepatic epithelial cells

C5a, a 74 amino acid peptide cleaved from the complement protein C5, is an extremely potent anaphylatoxin. Expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is also expressed in hepatocytes as well as in pulmonary epithelial, endothelial and smooth muscle cells. In the present study, we investigated the tissue distribution of C5aR by immunohistochemistry in normal human lung, liver, intestine and kidney using well‐defined monoclonal antibodies (mAbs) directed against the extracellular N‐terminus of the receptor. In all tissues examined, macrophages displayed an abundant expression of C5aR protein. However, in the normal human lung, C5aR expression was not detectable in bronchial and alveolar epithelial cells or in vascular smooth muscle or endothelial cells. In the normal human liver, no C5aR protein was detected in hepatocytes, whereas Kupffer cells strongly expressed the C5aR. In normal human kidney, the C5aR was detectable only in proximal tubular cells. C5aR gene transcription in Kupffer cells and proximal tubular cells was confirmed by in situ hybridization. Thus, our results point to an as yet unknown role of the C5aR in normal renal physiology. In the normal lung and liver, however, previous evidence for the ubiquitous expression of C5aR in epithelial, endothelial and smooth muscle cells
in situ should be re‐evaluated.

Histone deacetylase inhibitor valproic acid inhibits cancer cell proliferation via down-regulation of the alzheimer amyloid precursor protein.

The β-amyloid precursor protein (APP) represents a type I transmembrane glycoprotein that is ubiquitously expressed. In the brain, it is a key player in the molecular pathogenesis of Alzheimer disease. Its physiological function is however less well understood. Previous studies showed that APP is up-regulated in prostate, colon, pancreatic tumor, and oral squamous cell carcinoma. In this study, we show that APP has an essential role in growth control of pancreatic and colon cancer. Abundant APP staining was found in human pancreatic adenocarcinoma and colon cancer tissue. Interestingly, treating pancreatic and colon cancer cells with valproic acid (VPA, 2-propylpentanoic acid), a known histone deacetylase (HDAC) inhibitor, leads to up-regulation of GRP78, an endoplasmic reticulum chaperone immunoglobulin-binding protein. GRP78 is involved in APP maturation and inhibition of tumor cell growth by down-regulation of APP and secreted soluble APPα. Trichostatin A, a pan-HDAC inhibitor, also lowered APP and increased GRP78 levels. In contrast, treating cells with valpromide, a VPA derivative lacking HDAC inhibitory properties, had no effect on APP levels. VPA did not modify the level of epidermal growth factor receptor, another type I transmembrane protein, and APLP2, a member of the APP family, demonstrating the specificity of the VPA effect on APP. Small interfering RNA-mediated knockdown of APP also resulted in significantly decreased cell growth. Based on these observations, the data suggest that APP down-regulation via HDAC inhibition provides a novel mechanism for pancreatic and colon cancer therapy.

Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis.

Immunity against mycobacteria is almost exclusively confined to epithelioid cell granulomas, where a long‐lasting but labile balance exists between host and bacilli. The relationship between immunity and mycobacteria results in regression, growth, or caseation of granulomas. To prove whether caseation is associated with apoptosis, biopsy specimens of patients with tuberculosis were analysed by electron microscopy and by in situ end‐labelling combined with immunofluorescence. Apoptotic cells were not detected in regressive granulomas. Whereas productive granulomas without histologically recognizable caseous necrosis revealed only single apoptotic cells, large numbers of apoptotic CD68+ macrophages and apoptotic CD3+, CD45RO+ T cells were observed within caseous foci. As prime candidates undergoing and/or eliciting apoptosis, vital cells surrounding caseous foci were characterized. Immunohistochemistry showed that the majority of vital CD68+ macrophages surrounding caseous foci are negative for the anti‐apoptotic protein bcl2, but positive for the pro‐apoptotic protein bax. In situ hybridization combined with immunofluorescence demonstrated that the majority of the adjacent lymphocytes are activated CD3+, CD45RO+ cells expressing the pro‐inflammatory cytokine interferon γ (IFNγ) and the death ligand FasL. These results suggest that caseation is strongly associated with apoptosis of macrophages and T lymphocytes; that the onset of apoptosis in macrophages may be promoted by the lack of bcl2 and the abundance of bax; and that activation‐induced cell death (AICD) may be responsible for the apoptosis of T cells. Copyright

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.

Blockade of the type I IGF receptor expression in human prostate cancer cells inhibits proliferation and invasion, up-regulates IGF binding protein-3, and suppresses MMP-2 expression

The type I insulin‐like growth factor receptor (IGF‐IR) is involved in tumour cell proliferation, invasion, and cancer cell survival. Several studies indicate that the IGF axis contributes to prostate cancer pathogenesis, but there is no consensus regarding the relative expression of the IGF‐IR in benign and malignant prostate epithelium. In this study, endogenous IGF‐IR gene expression was reduced in stably transfected PC‐3 cells by employing an antisense RNA strategy which resulted in significant suppression of both PC‐3 cell invasion and proliferation in vitro. Furthermore, it was demonstrated that a direct correlation exists between the inhibition of IGF‐IR gene expression and either up‐regulation of IGF binding protein (BP)‐3 or down‐regulation of matrix metalloproteinase (MMP)‐2 expression in androgen‐independent PC‐3 cells. Moreover, inhibition of IGF‐IR gene expression in transfected PC‐3 cells leads to an enhanced rate of spontaneous apoptosis. In addition, expression analyses by quantitative RT‐PCR on RNA from laser microdissected matched normal prostate and prostate tumour samples revealed that IGF‐IR gene expression was up‐regulated in nine of 12 prostate cancers, whereas IGFBP‐3 gene expression was down‐regulated in all 12 prostate carcinomas analysed. These results indicate an important role for IGF‐IR and IGFBP‐3 in the homeostasis of prostate carcinoma cells and provide a further basis for targeting IGF‐IR or IGFBP‐3 gene expression in order to improve understanding of the IGF‐IR‐activated signalling pathways and as a potential treatment for prostate cancer. Copyright

Bax Inhibitor-1 Is Overexpressed in Prostate Cancer and Its Specific Down-Regulation by RNA Interference Leads to Cell Death in Human Prostate Carcinoma Cells

To analyze differential gene expression of putative prostate tumor markers we compared the expression levels of more than 400 cancer-related genes using the cDNA array technique in a set of capsule-invasive prostate tumor and matched normal prostate tissue. The overexpression of Bax inhibitor-1 (BI-1) in prostate carcinoma and prostate cancer cell lines was confirmed by using Northern blot and Western blot analyses. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) on intact RNAs from 17 paired laser-captured microdissected epithelial tissue samples confirmed up-regulated BI-1 expression in 11 of 17 prostate tumors. In addition, it was demonstrated that BI-1 expression is down-regulated in stromal cells as compared to matched normal epithelial cells of the prostate. In situ hybridization experiments on prostate sections also revealed that BI-1 expression is mainly restricted to epithelial cells. Furthermore, quantitative RT-PCR on RNAs derived from five benign prostate hyperplasia (BPH) samples showed no significant difference in BI-1 expression as compared to normal epithelial prostate tissue. To determine the function of BI-1 in vitro, human PC-3, LNCaP, and DU-145 prostate carcinoma cells were transfected with small interfering double-strand RNA (siRNA) oligonucleotides against the BI-1 gene leading to a specific down-regulation of BI-1 expression. Furthermore, transfection of PC-3, LNCaP, and DU-145 cells with BI-1 sequence-specific siRNAs caused a significant increase in spontaneous apoptosis in all cell lines. Taken together, our results indicate that the human BI-1 gene contains the potential to serve as a prostate cancer expression marker and as a potential target for developing therapeutic strategies for prostate cancer.

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 and functional analysis of Bax inhibitor-1 in human breast cancer cells.

Recently, deregulated expression of the anti‐apoptotic protein Bax inhibitor‐1 (BI‐1) has been shown in several human cancers. In this report, we show that BI‐1 is expressed at various levels in six different human breast cancer cell lines. In order to investigate the function of BI‐1 in oestrogen‐dependent MCF‐7, T‐47D and oestrogen‐independent MDA‐MB‐231 breast cancer cells, the RNA interference technique was used to knock down BI‐1 expression specifically. Suppression of BI‐1 expression caused a significant increase in spontaneous apoptosis in MDA‐MB‐231 cells, whereas MCF‐7 and T‐47D cells remained almost unaffected. Furthermore, BI‐1 expression analysis using a cancer profiling array showed up‐regulation of BI‐1 expression in cancer samples of breast, uterus and ovary, whereas down‐regulated BI‐1 expression was identified in stomach, colon, kidney, lung and rectal cancer. In addition, immunohistochemical studies using a BI‐1‐specific antibody on human breast cancer specimens also revealed that BI‐1 is expressed in the majority of cases. Moreover, to analyse whether BI‐1 expression is oestrogen receptor‐dependent, tumour cells were treated with oestradiol, ICI and tamoxifen: this showed no significant changes in BI‐1 expression. Taken together, our results demonstrate that BI‐1 expression is differentially deregulated in different cancers and that BI‐1 plays an important role in preventing certain breast cancer cells from undergoing apoptosis. Thus, the development of novel therapeutic strategies based on targeting BI‐1 gene expression in breast cancer could be restricted to selected individual cancer types. Copyright

C5a Receptor and Interleukin-6 Are Expressed in Tissue Macrophages and Stimulated Keratinocytes but not in Pulmonary and Intestinal Epithelial Cells

The anaphylatoxin derived from the fifth component of the human complement system (C5a) mediates its effects by binding to a single high-affinity receptor (C5aR/CD88), the expression of which has been traditionally thought to be restricted to granulocytes, monocytes, macrophages (Mphi), and cell lines of myeloid origin. Recent immunohistochemical data suggested that human bronchial and alveolar cells express C5aR as well. To reexamine the tissue distribution of human C5aR expression, transcription of the C5aR gene was investigated in normal and pathologically affected human lung (bronchopneumonia, tuberculosis), large intestine (acute appendicitis, Crohns disease), and skin (pyogenic granuloma, lichen planus) using in situ hybridization. In contrast to previous evidence, C5aR mRNA could not be detected in pulmonary or intestinal epithelial cells, whereas keratinocytes in inflamed but not in normal skin revealed detectable levels of C5aR transcripts. Additionally, it could be documented that only migrating Mphi express C5aR mRNA, whereas sessile Mphi in normal tissues and epithelioid/multinucleated Mphi found in granulomatous lesions do not. Because C5a has been demonstrated to upregulate the expression of interleukin (IL)-6 in human monocytes, we also studied IL-6 gene transcription in parallel to the C5aR. IL-6 mRNA was detectable in many tissue Mphi. Surprisingly, a tight co-expression of C5aR and IL-6 mRNA was observed in keratinocytes from lesions of pyogenic granuloma and lichen planus. These results point to an as yet unknown role for C5a in the pathogenesis of skin disorders beyond its well-defined function as a chemoattractant and activator of leukocytes.

Cyclopamine treatment of full‐blown Hh/Ptch‐associated RMS partially inhibits Hh/Ptch signaling, but not tumor growth

Mutations in the Hedgehog (Hh) receptor Patched (Ptch) are responsible for a variety of tumors, which show ligand‐independent stimulation of the Hh/Ptch signaling cascade. Cyclopamine is an alkaloid of the corn lily Veratrum californicum, which blocks activity of the pathway by inhibition of Smoothened (Smo), the signal transduction partner of Ptch. This results in growth inhibition of Hh/Ptch‐dependent tumor cells in vitro, of subcutaneous xenografts as well as of precancerous lesions in Ptch+/− mice. However, the evidence that treatment with cyclopamine is an effective anti‐cancer therapy against full‐blown tumors is sparse. Here, we have investigated the responsiveness of full‐blown Hh/Ptch‐associated rhabdomyosarcoma (RMS) to this drug. Hh pathway activity and proliferation of cultured primary RMS cells was inhibited by cyclopamine. Hh signaling was also partially suppressed by the drug in RMS in vivo, but cyclopamine treatment did not result in stable disease or tumor regression. It also did not affect proliferation, apoptosis or the differentiation status of RMS. This was in contrast to anti‐proliferative effects on tumor growth caused by doxorubicin, an anthracycline routinely used in therapy of human RMS. In summary, our data indicate that there must be additional factors that render full‐blown Hh/Ptch‐associated RMS insensitive against anti‐proliferative effects of cyclopamine in vivo.