Hans-Gerd Pauels

IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP‐1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL‐4, IL‐10 and IL‐13 have been described to exert anti‐inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP‐1 by activated intestinal epithelial cells. We examined Caco‐2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP‐1 was determined under stimulated and non‐stimulated conditions. IL‐4, IL‐10 and IL‐13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL‐1β or tumour necrosis factor‐alpha (TNF‐α), we observed markedly increased concentrations of MCP‐1 in supernatants of Caco‐2 cells and intestinal epithelial cells. IL‐4, IL‐10 and IL‐13 all had the capacity to down‐regulate the production of MCP‐1 in Caco‐2 cells as well as in freshly isolated epithelial cells. Caco‐2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL‐1β or TNF‐α for MCP‐1 production. As MCP‐1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP‐1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.

Role of the CD95/CD95 ligand system in glucocorticoid-induced monocyte apoptosis.

Glucocorticoids (GC) act as potent anti-inflammatory and immunosuppressive agents on a variety of immune cells. However, the exact mechanisms of their action are still unknown. Recently, we demonstrated that GC induce apoptosis in human peripheral blood monocytes. In the present study, we examined the signaling pathway in GC-induced apoptosis. Monocyte apoptosis was demonstrated by annexin V staining, DNA laddering, and electron microscopy. Apoptosis required the activation of caspases, as different caspase inhibitors prevented GC-induced cell death. In addition, the proteolytic activation of caspase-8 and caspase-3 was observed. In additional experiments, we determined the role of the death receptor CD95 in GC-induced apoptosis. CD95 and CD95 ligand (CD95L) were up-regulated in a dose- and time-dependent manner on the cell membrane and also released after treatment with GC. Costimulation with the GC receptor antagonist mifepristone diminished monocyte apoptosis as well as CD95/CD95L expression and subsequent caspase-8 and caspase-3 activation. In contrast, the caspase inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde suppressed caspase-3 activation and apoptosis, but did not down-regulate caspase-8 activation and expression of CD95 and CD95L. Importantly, GC-induced monocyte apoptosis was strongly abolished by a neutralizing CD95L mAb. Therefore, our data suggest that GC-induced monocyte apoptosis is at least partially mediated by an autocrine or paracrine pathway involving the CD95/CD95L system.

IL‐10 induces apoptosis in human monocytes involving the CD95 receptor/ligand pathway

The cytokine IL‐10 exerts potent immunosuppressive and anti‐inflammatory effects, although the mechanisms of this action remain largely unknown. In the present study, we investigated the effects of IL‐10 in human peripheral blood monocytes. We were able to demonstrate that IL‐10 dose‐ and time‐dependently triggers apoptosis in these cells as detected by annexin‐V staining, the nick end labeling (TUNEL) procedure, electron microscopy and analysis of DNA laddering. IL‐10‐induced apoptosis required the activation of proteases of the caspase family, since a peptide caspase inhibitor attenuated cell death and, in addition, the proteolytic activation of caspase‐8 was observed. Since caspase‐8 has been implicated as a regulator of apoptosis mediated by death receptors, we investigated a potential involvement of the CD95 receptor/ligand system. Indeed, treatment of monocytes with IL‐10 induced a dose‐dependent up‐regulation of CD95 receptor and ligand expression on the monocyte surface. Furthermore, a CD95 ligand‐neutralizing antibody significantly inhibited IL‐10‐induced apoptosis. In summary, our data show that IL‐10 triggers monocyte apoptosis involving the CD95 system via an autocrine or paracrine process. Therefore, at least part of the anti‐inflammatory properties of IL‐10 may involve induction of apoptosis in monocytes.

Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-γ) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co‐culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co‐incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco‐2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three‐colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti‐CD4, anti‐CD8, anti‐IFN‐γ and anti‐IL‐4. Patients with IBD (Crohns disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co‐incubation with Caco‐2 cells we found a significant increase of IFN‐γ‐producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN‐γ+/CD8+ lymphocytes in patients with UC was also seen after direct co‐incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I‐restricted. No significant epithelial cell‐mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN‐γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN‐γ‐producing CD8+ lymphocytes.

Down-Regulation of Monocyte Apoptosis by Phagocytosis of Platelets: Involvement of a Caspase-9, Caspase-3, and Heat Shock Protein 70-Dependent Pathway

Monocytes interact and cross-talk with platelets in many settings including inflammation, hemostasis, or vascular disorders. During inflammatory diseases, there is a rapid targeting of monocytes and platelets to points of inflammation and endothelial injury, where they lie side-by-side. In this in vitro study, we investigated different interactions between monocytes and platelets and elucidated whether platelets might affect monocyte apoptosis. Freshly isolated human monocytes were rendered apoptotic by serum deprivation or CD95 ligation and cocultured with platelets. Monocyte apoptosis was determined by flow cytometry, TUNEL staining, DNA electrophoresis, and transmission electron microscopy imaging. We could show that monocyte apoptosis was highly suppressed when platelets were added to the cultures. Transmission electron microscopy depicted that monocytes completely ingested thrombocytes by phagocytosis. Blocking thrombocyte uptake by the phagocytosis inhibitor cytochalasin D abrogated the enhanced monocyte survival and led to high apoptosis levels. Monocyte survival was paralleled by down-regulation of caspase-9 and -3 and up-regulation of heat shock protein 70 during uptake of platelets. Platelet supernatants and contents of platelet granules were ineffective in altering monocyte senescence. Also, ingestion of latex beads or zymosan by monocytes was ineffective to mimic platelet-dependent rescue from apoptosis. In conclusion, this study shows that platelets can suppress apoptosis of monocytes by a specific phagocytosis-dependent process with further consequences for atherosclerotic or inflammatory conditions.

Prostaglandins, but not tumor-derived IL-10, shut down concomitant tumor-specific CTL responses during murine plasmacytoma progression

IL‐10 is assumed to be a major immunosuppressive factor produced by most B‐cell tumors. The immunosuppressive role of tumor‐derived IL‐10 was analyzed using the MHC class II‐negative BALB/c plasmacytoma ADJ‐PC‐5 as a model tumor. Immune monitoring of tumor‐bearing mice was based on the measurement of tumor burden, tumor‐specific CTL cytotoxicity and intracellular cytokine staining using FACS. ADJ‐PC‐5 tumor progression in syngeneic recipients is associated with strong, concomitant, tumor‐specific CTL responses during early stages of tumor progression which are sufficient to cause rejection of small s.c. autologous test tumors. These initial CTL responses gradually decline during later tumor stages. Blocking of IL‐10 in vivo did not abolish CTL suppression or retard tumor growth. More strikingly, application of anti‐IL‐10 antibodies during early tumor stages abrogated CTL induction and markedly accelerated tumor growth. In contrast to anti‐IL‐10 treatment, application of cyclo‐oxygenase inhibitors to ADJ‐PC‐5 tumor‐bearing mice led to enhanced tumor‐specific CTL responses throughout all stages of tumor progression, paralleled by retarded tumor growth and a significantly delayed onset of suppression. Both findings contradict a dominant immunosuppressive role of IL‐10 during B‐cell tumor progression. Tumor‐derived IL‐10 must therefore be considered an immunostimulating factor, which accounts for the high immunogenicity of B‐cell tumors, whereas prostaglandins, which are not produced by the tumor cells themselves, are the dominant immunosuppressors in this system.

Protein kinase C (PKC) dependent induction of tissue factor (TF) by mesangial cells in response to inflammatory mediators and release during apoptosis

In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin‐1 (IL‐1β), tumour necrosis factor alpha (TNF‐α) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. IL‐1β, TNF‐α and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA‐AM, a chelator of intracellular [Ca2+]i as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down‐regulated cytokine‐induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL‐1β did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL‐1β treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. Inflammatory mediators up‐regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed‐back link. Apoptosis of inflammatory MC may trigger to spread PCA.

Suppression of Tumour-Specific Cytotoxic T-Cell Responses Against the Syngeneic BALB/c Plasmacytoma ADJ-PC-5 by Tumour-Induced CD8+ Regulatory T Cells Via IFN-γ

The mechanisms of tolerance induction by tumour cells during early stages of tumourigenesis were analysed in a murine model system using the highly immunogenic BALB/c plasmacytoma ADJ‐PC‐5. Early stages of tumourigenesis were simulated in syngeneic BALB/c mice by repeated intraperitoneal injections with subimmunogenic doses of X‐irradiated ADJ‐PC‐5 tumour cells. This treatment causes a state of tumour‐specific tolerance in a high percentage of mice, involving a population of CD8+ peritoneal T cells which are able to suppress a protective tumour‐specific Tc response against this tumour. Using a primary mixed lymphocyte tumour cell culture (MLTC) as an in vitro system to study suppressive mechanisms of such regulatory T cells, the role of production or consumption of a number of cytokines was analysed. The data presented here demonstrate that inhibition of a protective Tc response against ADJ‐PC‐5 tumour cells is due to IFN‐γ production by suppressive T cells from tolerized mice, but not to IL‐2 consumption. In contrast to typical CD8+ Tc cells, ADJ‐PC‐5‐specific CD8+ Tc cells do not produce IFN‐γ and are furthermore suppressed by IFN‐γ. Thus, tumour‐induced suppressive T cells and tumour‐specific Tc cells seem to represent functionally and phenotypically different subsets of CD8+ T cells, possibly pointing towards a differential activation of type‐1 and type‐2 CD8+ T cells depending on the dose of tumour cells.

Differential Activation of CD8+ Tumor-Specific Tc1 and Tc2 Cells by an IL-10-Producing Murine Plasmacytoma

The involvement of counteractive CD8+ T-cell subsets during tumor-specific immune responses was analyzed in a syngeneic murine plasmacytoma model. CD8+ Tc cells against the immunogenic IL-10-producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced by immunization of BALB/c mice with X-irradiated ADJ-PC-5 tumor cells in vivo and in vitro. However, the failure of recipient mice to mount a protective Tc response against the tumor during early stages of a real or simulated tumor growth is not due to immunological ignorance, but depends on the induction of tumor-specific tolerance, involving a population of tumorinduced CD8+ T cells that are able to inhibit the generation of tumor-specific Tc cells in a primary ADJ-PC-5-specific MLTC, using IFN-γ as a suppressive factor. Whereas most longterm cultivated CD8+ ADJ-PC-5-specific Tc lines produce type-1 cytokines on stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum. Furthermore, the primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a Tc2 response. The Tc response is strictly depending on tumor-derived IL-10. CD8+ Tc cells that are induced in a primary MLTC do not produce IFN-γ, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-γ or IL-12. In contrast, ADJ-PC- 5-specific CD8+ Tc cells from immunized mice are IFN-γ producing Tc1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by the smallest numbers of irradiated ADJ-PC-5-specific Tc1 cells via IFN-γ these Tc1 cells behave similar to the suppressive CD8+ T cells that are induced during early stages of ADJ-PC-5 tumorigenesis.

CD8+ tumor-specific Tc cells primed in vivo or in vitro against the BALB/c plasmacytoma ADJ-PC-5 use the same TcR V#x03B2; families but display distinct TC1 or TC2 characteristics

The involvement of counteractive CD8+ T cell subsets in tumor-specific unresponsiveness was analyzed in a syngeneic murine tumor model. CD8+ cytotoxic T cells against the IL-10 producing BALB/c plasmacytoma ADJ-PC-5 can be easily induced in vitro, in a primary syngeneic mixed lymphocyte tumor cell culture (MLTC), or in vivo, by repeated immunization of syngeneic BALB/c mice with high doses of X-irradiated ADJ-PC-5 tumor cells. Long term cultivated CD8+ ADJ-PC-5-specific Tc lines use either TcR of the V beta 6 or V beta 8.1/8.2 type, irrespective if the lines were derived from a primary MLTC or from immunized mice. While most of the Tc lines produce type-1 cytokines (IFN-gamma, no IL-4) upon stimulation, at least two of them, which were derived from a primary MLTC, display a type-2 cytokine spectrum (IL-4, no IFN-gamma). The primary in vitro Tc response against ADJ-PC-5 cells shows characteristics of a TC2 response: CD8+ Tc cells which are induced in a primary MLTC do not produce IFN-gamma, and the tumor-specific Tc response is enhanced by IL-4 but suppressed by IFN-gamma or IL-12. In contrast, ADJ-PC-5-specific CD8+ Tc cells from immunized mice are IFN-gamma producing TC1 cells. Since the primary in vitro Tc response against the tumor is suppressed even by lowest numbers of irradiated ADJ-PC-5-specific TC1 cells via IFN-gamma, these TC1 cells behave similar to a previously described regulatory subset of IFN-gamma producing CD8+ T cells, which are induced during early stages of ADJ-PC-5 tumorigenesis and inhibit the induction of a tumor-specific Tc response from naive BALB/c spleen cells in vitro.