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Martin Herrmann

Publications and source records attributed to Martin Herrmann.

34 records · Page 2Linked to original sources
PubMed2005

Extravasation into synovial tissue induces CCL20 mRNA expression in polymorphonuclear neutrophils of patients with rheumatoid arthritis.

OBJECTIVE: Examination of expression of the chemokine macrophage inflammatory protein-3a (CCL20/Mip-3alpha) in blood polymorphonuclear neutrophils (PMN) and synovial fluid (SF) PMN of patients with rheumatoid arthritis (RA). METHODS: Paired samples of blood PMN and SF PMN were obtained from 11 patients with RA. In addition, SF was prepared from 9 patients with osteoarthritis (OA) and 10 patients with juvenile idiopathic arthritis (JIA). PMN were isolated via density centrifugation to a purity of 98%. Total RNA was isolated and the expression of CCL20 was determined by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In some experiments blood PMN obtained from healthy donors were stimulated with individual SF of patients with RA. For quantitative considerations, CXCL8, CCL20, interleukin 1, and tumor necrosis factor-alpha (TNF-alpha) levels were determined in SF by ELISA. RESULTS: In SF of RA patients CCL20 and CXCL8 levels were elevated, up to 7.5 ng/ml and 23.6 ng/ml, respectively. No significant level of either chemokine was found in SF of patients with JIA and OA. CCL20 mRNA was undetectable in blood PMN of all patients with RA. In SF PMN, CCL20 mRNA was found in 6/11 RA patients. Expression of CCL20 mRNA in 5/6 SF PMN samples was observed in the absence of detectable TNF-alpha levels in SF. Cell culture experiments, however, confirmed the ability of TNF-alpha in SF to induce CCL20 mRNA expression in blood PMN. Notably, expression of CCL20 was also found in PMN after stimulation with SF lacking TNF-alpha. CONCLUSION: Recruitment of PMN to the synovial microenvironment induces expression of CCL20 mRNA independent of the concentrations of TNF-alpha accumulating in SF of patients with RA.

Adult↗
PubMed2004

Impaired clearance of dying cells in systemic lupus erythematosus.

Impaired clearance of apoptotic cell material has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Besides many other molecules, C1q and DNaseI contribute to an efficient clearance of dying cells. A frequently observed factor in SLE patients is the accumulation of unusually large amounts of apoptotic cells in various tissues. We showed that in a subgroup of patients with SLE, apoptotic cells accumulated in the germinal centers of the lymph nodes. The numbers of tingible body macrophages usually containing engulfed apoptotic nuclei were significantly reduced in these patients. Furthermore, we differentiated macrophages from CD34+ stem cells of SLE patients and NHD in vitro to analyze whether the observed clearance defects are intrinsic. Indeed, macrophages from SLE patients showed a reduced phagocytic capability. Very interestingly, those macrophages from different SLE patients, as well as granulocytes from these patients, showed in part different phagocytic defects, suggesting a heterogeneous clearance defect. We conclude that a failure of clearance in the early phase of apoptosis leads to a secondary necrotic status of the cells. Danger signals are released, modified autoantigens are accessible, and an autoimmune reaction gets started.

Animals↗
PubMed2004

Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo.

Strategies to enhance the immunogenicity of tumors are urgently needed. Although vaccination with irradiated dying lymphoma cells recruits a tumor-specific immune response, its efficiency as immunogen is poor. Annexin V (AxV) binds with high affinity to phosphatidylserine on the surface of apoptotic and necrotic cells and thereby impairs their uptake by macrophages. Here, we report that AxV preferentially targets irradiated lymphoma cells to CD8+ dendritic cells for in vivo clearance, elicits the release of proinflammatory cytokines and dramatically enhances the protection elicited against the tumor. The response was endowed with both memory, because protected animals rejected living lymphoma cells after 72 d, and specificity, because vaccinated animals failed to reject unrelated neoplasms. Finally, AxV-coupled irradiated cells induced the regression of growing tumors. These data indicate that endogenous adjuvants that bind to dying tumor cells can be exploited to target tumors for immune rejection.

Animals↗
PubMed2004

Cooperation between C1q and DNase I in the clearance of necrotic cell-derived chromatin.

OBJECTIVE: The efficient uptake of dying cells by phagocytes is essential to the avoidance of chronic inflammation. Some human autoimmune responses are thought to be driven by autoantigens from apoptotic or necrotic cells. We analyzed the role of C1q and DNase I in the disposal of necrotic cell-derived chromatin because deficiencies in these serum factors predispose to the development of systemic autoimmune disorders, such as systemic lupus erythematosus. METHODS: Human necrotic peripheral blood lymphocytes were incubated in cell culture medium supplemented with various sera or serum components. Chromatin degradation was monitored by measuring the residual DNA content by flow cytometry. The uptake of necrotic cell-derived nuclei was analyzed by in vitro phagocytosis assays. RESULTS: Reconstitution of C1q-depleted serum with C1q strongly increased its ability to degrade necrotic cell-derived chromatin. Although C1q itself displayed no DNase activity, it strongly augmented the activity of serum DNase I. Whereas an excess of recombinant DNase I degraded chromatin in the absence of C1q, efficient uptake of the predigested material by monocyte-derived phagocytes required the presence of C1q. These data show that C1q and DNase I cooperate in the degradation of chromatin from necrotic cells. Furthermore, C1q was found to be necessary for effective uptake of degraded chromatin by monocyte-derived phagocytes. CONCLUSION: C1q or DNase I deficiencies may precipitate autoimmunity in humans by a mechanism similar to that of other molecules that are involved in the safe, efficient, and rapid disposal of dying cells.

Apoptosis↗
PubMed2004

Loss of GM1 surface expression precedes annexin V-phycoerythrin binding of neutrophils undergoing spontaneous apoptosis during in vitro aging.

BACKGROUND: Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation. Apoptotic neutrophils undergo specific alterations in their receptor profiles. These alterations are likely to contribute to the characteristic functional silencing of the dying cells. METHODS: By flow cytometry and fluorescence microscopy, we analyzed the ganglioside GM1, a lipid raft marker, with respect to its surface expression on neutrophil and eosinophil granulocytes. Apoptosis was monitored by morphological changes and by the binding of annexin V-phycoerythrin (AxV-PE). RESULTS: GM1, which was stained by the cholera toxin subunit B, was found only on neutrophil granulocytes; eosinophil granulocytes did not bind cholera toxin subunit B. GM1 was lost from the surfaces of neutrophils before AxV-PE binding (early apoptosis). Surprisingly, GM1 reappeared during the late stages of apoptosis, although without functional consequences. GM1 was found on the cell surface and in intracellular membranes, whereas CD16 was found only at the cell surface. CONCLUSIONS: Loss of surface GM1 is a new marker for the detection of the aging of neutrophils. Its loss precedes the binding of AxV-PE of neutrophils.

Annexin A5↗
PubMed2004

The induction of TGF-beta(1) and NF-kappaB parallels a biphasic time course of leukocyte/endothelial cell adhesion following low-dose X-irradiation.

BACKGROUND AND PURPOSE: Low-dose radiotherapy (LD-RT) is known to exert an anti-inflammatory effect, but the knowledge of the underlying molecular mechanisms is still scarce. The authors have recently reported that transforming growth factor beta 1 (TGF-beta(1)) essentially contributes to a reduced endothelial adhesion of mononuclear cells (PBMC) following LD-RT. Furthermore, TGF-beta(1) secretion was associated with the induction of the transcription factor nuclear factor kappa B (NF-kappaB). However, the time course of adhesion, TGF-beta(1) expression, and NF-kappaB activity following LD-RT has not been thoroughly investigated yet. MATERIAL AND METHODS: The human EA.hy.926 endothelial cell line (EA.hy.926 EC) was grown to 95% confluence. Immediately after stimulation with the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha), EA.hy.926 EC were irradiated with single doses ranging from 0.3 up to 3 Gy. Adhesion assays were performed 4, 12, and 24 h after irradiation. Nuclear extracts and culture supernatants were harvested after 4, 8, 12, 20, 24, 30, and 40 h. NF-kappaB DNA-binding activity was analyzed by electrophoretic mobility shift assays (EMSA) and TGF-beta(1) secretion by enzyme-linked immunosorbent assay (ELISA). The functional impact of TGF-beta(1) on the course of leukocyte/EC adhesion was analyzed by neutralizing TGF-beta(1) in parallel with stimulation/irradiation of the EA.hy.926 EC. RESULTS: 4 and 24 h after irradiation of EA.hy.926 EC in the dose range between 0.3 and 0.7 Gy, a reduced adhesion of PBMC compared to nontreated controls could be observed. However, 12 h after irradiation a relative maximum of adhesion (up to 30% increase) was seen at a dose of 0.3 Gy. TGF-beta(1) secretion and NF-kappaB DNA-binding activity displayed a similar biphasic kinetics of induction with a relative minimum 12 h after irradiation. Neutralization of TGF-beta(1) activity restored adhesion at 4 and 24 h after LD-RT of EA.hy.926 EC, but it did not influence leukocyte adhesion 12 h after irradiation. CONCLUSION: LD-RT of stimulated human EA.hy.926 EC is followed by a biphasic time course of NF-kappaB activity and an increased secretion of TGF-beta(1). The kinetics shows peak levels at 4-8 h and 24-30 h after LD-RT and results in a biphasic leukocyte/EC adhesion profile.

Cell Adhesion↗
PubMed2004

Defects in the disposal of dying cells lead to autoimmunity.

The fast and efficient uptake of dying cells is of main importance to prevent contact of the immune system with intracellular autoantigens. Insufficient clearance of the latter is discussed to drive the humoral autoimmune response in systemic lupus erythematosus. Many adaptor molecules and receptors are involved in the recognition of dying cells. In this paper we focus on the involvement of phosphatidylserine, glycoproteins, and complement and DNaseI in the clearance of apoptotic and necrotic cells, respectively. Furthermore, extracellular danger signals released from necrotic cells are discussed and the uptake process of primary necrotic cells is investigated in detail. Last but not least, the character and origin of clearance defects observed in some systemic lupus erythematosus patients is presented.

Autoimmunity↗
PubMed2004

Apoptosis and systemic lupus erythematosus.

Reduced clearance of dying cells by macrophages or increased apoptosis provokes accumulation of cellular fragments in various tissues. This process seems to induce the uptake of autoantigens from apoptotic nuclei or chromatin by dendritic cells (DCs). Then, the DCs present altered self-epitopes to naive T cells. Thus, autoreactive T cells are activated accidentally and may now provide T-cell help for B cells that present peptides processed from secondary necrotic/late apoptotic prey. Impaired phagocytic removal of early apoptotic cells may cause accumulation of secondary necrotic cells and debris in the germinal centers of secondary lymph organs. The latter bind complement and can, therefore, be trapped on the surfaces of follicular DCs (FDCs). B cells may get in contact with intracellular autoantigens that had been released during late stages of apoptotic cell death and are immobilized by FDCs. Consecutively, B cells that had, for example, gained specificity for nuclear auto-antigens during random somatic mutations can receive a short-term survival signal. After migration into the mantle zone, these autoreactive B cells may finally be activated by autoreactive CD4+ T helper cells. B cells then differentiate into memory or plasma cells. The plasma cells produce those pathogenic nuclear autoantibodies. Many defects are known with respect to the clearance of apoptotic cells and cell material, especially that of nuclear origin. Reflecting on the plethora of defects of clearance of apoptotic material already demonstrated in systemic lupus erythematosus, it is reasonable to argue that, for many patients, failure of clearance is at the heart of their disease.

Apoptosis↗
PubMed2003

Polymorphonuclear granulocytes induce antibody-dependent apoptosis in human breast cancer cells.

Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.

Alleles↗
PubMed2003

Early detection of apoptosis by staining of acid-treated apoptotic cells with FITC-labeled lectin from Narcissus pseudonarcissus.

BACKGROUND: Exposure of anionic phospholipids and modified carbohydrates are main parts of the apoptotic death program. Cells undergoing apoptosis can be identified by various methods, detecting surface changes or modifications of their organelles, respectively. We describe a method for the detection of early apoptosis by staining of cells with fluorescein isothiocyanate (FITC)-labeled lectin from Narcissus pseudonarcissus (NPn). METHODS: Apoptosis in cells or in cell lines was induced by various stimuli. To detect apoptosis the cells were stained with FITC-labeled lectin of NPn. After a short-term acid treatment they were analyzed by flow cytometry. RESULTS: The instability of the cytoplasmic membrane against acid and the binding of NPn were very early features of apoptotic cell death. The NPn lectin staining procedure detected apoptosis with high sensitivity. The staining was stable for at least 12 h. CONCLUSIONS: The method described in this study is suitable for the detection of the very early phases of apoptosis. The NPn lectin staining after short-term acid treatment can, therefore, be added to the list of reliable tools for the research of cell death.

Acids↗
PubMed2003

UV or X-irradiation increases the cytoplasmic accumulation of rhodamine 123 in various cancer cell lines.

PURPOSE: Previous studies indicated that ATP-binding cassette (ABC) membrane transporters protect against UV-induced apoptosis. We investigated the effect of UVB and X-ray irradiation on the export function of these ABC transporters in primary lymphocytes and various cancer cell lines. MATERIAL AND METHODS: We used rhodamine accumulation assays in various human malignant cell lines and peripheral blood lymphocytes (PBL). Cells were irradiated with up to 960 mJ/cm2 and up to 50 Gy of UVB and X-ray, respectively. RESULTS: We demonstrated that UVB as well as X-ray irradiation inhibit the export function of the ABC transporters in a dose-dependent fashion. For PBL, this effect did not correlate with an apoptotic phenotype. In the case of the tumor cell lines, even though the irradiation-induced inhibition of membrane transporters was accompanied by phosphatidylserine exposure, only a minority of cells had lost their mitochondrial membrane potential during the observation period. Furthermore, we demonstrated that the inhibition of membrane transporters is not a general feature of apoptosis. CONCLUSION: Irradiation inhibits the export function of ABC transporters. Although some of the irradiated cells undergo apoptosis following irradiation, the inhibition is an unique feature accompanying irradiation and not a general hallmark of apoptotic cell death. The inhibition of drug export by irradiation may offer new potential for reverting multidrug resistance of cancer cells.

ATP-Binding Cassette Transporters↗
PubMed2003

Exposure of anionic phospholipids serves as anti-inflammatory and immunosuppressive signal--implications for antiphospholipid syndrome and systemic lupus erythematosus.

In contrast to necrotic cells, the clearance of apoptotic ones usually is an anti-inflammatory process which elicits only a marginal immune response. During apoptosis phosphatidylserine (PS) is exposed on the outer leaflet of the cytoplasmic membrane and serves as target for the PS receptor of phagocytes. The latter is responsible for anti-inflammatory signalling and the induction of TGFbeta. We were interested whether the immunogenicity of apoptotic cells can be increased by masking PS. We observed that treatment of xenogeneic apoptotic cells with annexin V (AxV) significantly increased the humoral immune response against surface epitopes of these cells. Furthermore, AxV-coated irradiated tumour cells were able to elicit a long lasting tumour specific cytotoxic T lymphocyte response. AxV efficiently blocked the uptake of irradiated cells by macrophages but not by dendritic cells. Furthermore, AxV skewed the phagocytosis of irradiated cells towards inflammation. Investigation of patients with autoimmune diseases further supported the role of anionic surface phospholipids for anti-inflammatory clearance of apoptotic cells. Impaired clearance and opsonisation with anti-phospholipid-antibodies are discussed to be responsible for the development of systemic lupus erythematosus and anti-phospholipid-syndrome, respectively. Presentation of cryptic epitopes from late apoptotic cells in a proinflammatory context may challenge T cell tolerance. In addition, accumulation of uncleared apoptotic debris in the germinal centres of lymph nodes may result in the survival of autoreactive B cells.

Animals↗
PubMed2002

MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.

BACKGROUND: Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126. METHODS: Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining. RESULTS: U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses. CONCLUSIONS: These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.

Annexin A5↗
PubMed2002

Impaired uptake of apoptotic cells into tingible body macrophages in germinal centers of patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the fate of apoptotic cells in the germinal centers (GCs) of patients with systemic lupus erythematosus (SLE). METHODS: Lymph node biopsy specimens obtained from 7 SLE patients with benign follicular hyperplasia, 5 non-SLE patients with benign follicular hyperplasia (non-SLE), 5 patients with malignant follicular lymphoma, and 3 patients with dermatopathic lymphadenitis were stained with monoclonal antibodies against macrophages (CD68) and follicular dendritic cells (CR2/CD21). TUNEL staining and transmission electron microscopy were performed to detect apoptotic cells. Confocal microscopy was used to evaluate the in vivo capacity of tingible body macrophages to remove apoptotic cell material. RESULTS: In a subgroup of patients with SLE, apoptotic cells accumulated in the GCs of the lymph nodes. The number of tingible body macrophages, which usually contained engulfed apoptotic nuclei, was significantly reduced in these patients. In contrast to what was observed in all controls, TUNEL-positive apoptotic material from SLE patients was observed to be directly associated with the surfaces of follicular dendritic cells (FDCs). CONCLUSION: Our findings suggest that in a sub-group of SLE patients, apoptotic cells are not properly cleared by tingible body macrophages of the GCs. Consequently, nuclear autoantigens bind to FDCs and may thus provide survival signals for autoreactive B cells. This action may override an important control mechanism for B cell development, resulting in the loss of tolerance for nuclear antigens.

Apoptosis↗