Naunyn-schmiedebergs Archives of Pharmacology, Jun 10, 2023
In previous studies, we demonstrated the involvement of H 4 R in inflammatory bowel disease (IBD)... more In previous studies, we demonstrated the involvement of H 4 R in inflammatory bowel disease (IBD) and IBD-associated colon cancer in mice and could ascribe H 4 R-mediated histamine function to colon epithelial cells. The transferability of obtained data to humans is however lacking. Functional expression of H 4 R on colon epithelial cells is a prerequisite to pursue the hypothesis of involvement of H 4 R in carcinogenesis. Thus, we here compared the expression of histamine receptor subtypes in a series of cell lines. Out of these, three colon-derived cell lines displaying different combinations of H 1 R and H 4 R expression were submitted to functional analyses. Human hematopoietic HMC-1, HL-60, and U937, lung-derived A549 and Calu-3, and colorectal LoVo, SW 480, Caco-2, HT-29, and HCT116 cells were included in the study. mRNA expression was quantified by RT-qPCR. For functional analyses, Caco-2, HT-29, and HCT116 cells were treated by incubation with 1-10 µM histamine in the presence or absence of selective histamine receptor antagonists. Calcium mobilization, cAMP accumulation, and cell proliferation were measured by fluorimetry, mass spectrometry, and real-time bioimpedance measurements, respectively. Histamine receptor expression was heterogeneous in the cell lines tested. In most cell lines, we detected H 1 R mRNA while H 4 R mRNAs were found only occasionally. The colon-derived epithelial cell lines LoVo, SW480, and HT-29 expressed H 1 R mRNA exclusively, while in HCT116 cells H 1 R and H 4 R mRNAs and in CaCo-2 H 2 R mRNA were detectable. Subsequent functional analyses in HT29, Caco-2, and HCT116 cells, however, indicated that only HT-29 responded to histamine stimulation, by means of H 1 R. For a detailed analysis of histamine receptor function, esp. that of H 1 R and H 4 R, in human colon-derived cell lines, the cell lines tested here are not fully convenient unless genetically modified.
Three cell surface molecules participate in lnterleukin-1 (IL-1) binding and signal generation, t... more Three cell surface molecules participate in lnterleukin-1 (IL-1) binding and signal generation, the two distinct types of receptors (type ! IL-IR and type II IL-IR) and the IL-1 receptor accessory protein 0L-1RAcP). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT-PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of IL-1 receptors was detected in all cell lines tested. In most cell lines the IL-IRAcP was co-expressed with the IL-I receptors, and only these lines responded to IL-I. However, in three cell lines no mRNA for the IL-IRAcP could be detected, and these cells did not respond to [L-I. These results suggest that the expression of the IL-IRAcP correlates with IL-I responsiveness and they point to a pivotal role for the IL-IRAcP in IL-I signal generation.
The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allerg... more The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allergic inflammation in the lungs. Besides their expression in leukocytes, receptors for C3a and C5a (C3aR and C5aR) have been noted in alveolar and bronchial epithelial cells, bronchial smooth muscle cells as well as in vascular endothelial and smooth muscle cells of normal and inflamed human and murine lungs. Recently, however, expression of anaphylatoxin receptors in parenchymal cells of the lung (and kidney) has been challenged. Using well-characterized monoclonal antibodies (mabs) against murine and rat anaphylatoxin receptors, we reexamined the pulmonary distribution of C3aR and C5aR. Immunohistochemistry was performed on frozen sections of lung tissues from normal mice and rats as well as from animals subjected to lipopolysaccharide (LPS)-induced inflammation or from MRL/lpr mice suffering from autoimmune disease. Furthermore, ovalbumin (OVA)-induced models of allergic asthma in the rat and mouse were investigated. Prominent expression of both anaphylatoxin receptors was detectable in resident as well as infiltrating leukocytes. No C3aR protein was observed in alveolar macrophages. Upon LPS-and OVA-challenge as well as in autoimmune inflammation, numbers of infiltrating leukocytes expressing prominent amounts of anaphylatoxin receptors increased. Even under these highly inflammatory conditions, however, expression of C3aR and C5aR was not inducible in parenchymal cells. Thus, our findings identify infiltrating leukocytes as a prominent source of anaphylatoxin receptors in inflamed lungs. A direct involvement of parenchymal cells in anaphylatoxin-mediated pulmonary inflammation is unlikely.
Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to f... more Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. autoMACS, gentleMACS, MACS, and MACSQuant are registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only.
Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, a... more Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, as detected by saturation binding and RT-PCR, and are responsive to IL-1beta activation by producing inflammatory cytokines IL-6 and IL-8. IL-1beta can also have an indirect effect on nervous cell functions, since it is able to modulate the stimulus-induced increase of intracellular Ca++ levels, one of the first steps of the cell activation mechanism. In fact, on SK-N-SH neuroblastoma cells, IL-1beta can inhibit the Ca++ increase induced by stimulation of acetylcholine receptors with carbachol. In parallel to IL-1beta, the neurotrophic factor CNTF also shows an inhibitory effect on carbachol-stimulated Ca++ increase in CNTFRalpha-expressing SK-N-SH cells. However, when simultaneously present, the two cytokines cross-inhibit, thus allowing full cell activation in response to the cholinoceptor agonist. The inhibitory effect of CNTF on IL-1beta activities on nervous cells was confirmed in th...
<p>MRL<sup>lpr</sup> mice displaying an IL-18 expression status as indicated we... more <p>MRL<sup>lpr</sup> mice displaying an IL-18 expression status as indicated were housed as described in the methods section. Individual ages (weeks) when they reached a health status ranking score ≥4 (<a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0140173#pone.0140173.t001" target="_blank">table 1</a>) were documented. Presented are pooled data from n = 23, 38, and 51 individuals of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> mice, respectively. The three data sets were analysed by Log-rank (Mantel-Cox) test and revealed no statistically significant differences.</p
<p>HEK293 mH<sub>1</sub>R cells (A+C) and HEK293 mH<sub>4</sub>R ce... more <p>HEK293 mH<sub>1</sub>R cells (A+C) and HEK293 mH<sub>4</sub>R cells (B+D) were incubated in the presence or absence of 50 ng/ml pertussistoxin (PTX) for 18 h. Changes in [Ca<sup>2+</sup>]<sub>i</sub> were determined as described in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g002" target="_blank">Figure 2</a> after stimulation with 100 µM histamine (HA) or 10 µM ATP as indicated on the x-axis (A+B). Intracellular cAMP concentrations were analyzed as described in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g003" target="_blank">Figure 3</a> after stimulation for 10 min with 100 µM forskolin (Forsk) and histamine (HA) (C+D). Forskolin-induced cAMP concentrations were 2385±160 pmol/mg protein in HEK293 H<sub>1</sub>R and 2164±436 pmol/mg protein in HEK 293 H<sub>4</sub>R cells, respectively. Data shown are means ± SD (n = 2, each consisting of 2–3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).</p
<p>Kidneys of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/... more <p>Kidneys of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> mice were removed, processed, and histologically analysed. (A) Presented are representative pictures of PAS-stained kidney sections obtained from MRL<sup>lpr</sup> (+/+), MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, (+/tm), and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> (tm/tm) mice. (B) Quantitative evaluations of the sections were performed using the scoring system as detailed in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0140173#pone.0140173.t002" target="_blank">Table 2</a>. Presented are individual data for three evaluated parameters from 5–11 mice per strain and the respective means +/- SD. Differences between the three strains were analysed by ANOVA with Bonferroni’s post test (*, p < 0.05; no indication: no significance).</p
Histamine is a pro-inflammatory mediator with a prominent role in allergic diseases. Antagonists ... more Histamine is a pro-inflammatory mediator with a prominent role in allergic diseases. Antagonists at the histamine receptor subtype 1 are central in anti-allergic therapies, with the exception of allergic asthma, where they are clinically without effect. The latest identified histamine receptor subtype 4, which is expressed mainly in hematopoietic cells, now provides a reasonable target for new therapeutic strategies inhibiting histamine function. The pathophysiology of allergy, esp. allergic asthma, and in its context the effects of antagonists at the histamine receptor subtype 4 in preclinical and clinical settings are discussed in this chapter.
... In genetic immunisation studies it was shown that far less DNA has to be applied by gene-gun ... more ... In genetic immunisation studies it was shown that far less DNA has to be applied by gene-gun as compared to needle injection, to obtain the same level of expression of the exogenous gene (Barry and Johnston, 1997; Feltquate et al, 1997; Pertmer et al, 1995). ...
Idiotype The term idiotype indicates the individuality of a particular BCR or TCR. It consists of... more Idiotype The term idiotype indicates the individuality of a particular BCR or TCR. It consists of a collection of conformational antigenic determinants (idiotopes) which are located within the combined variable domains of antibody H chains and L chains (F V fragment) or the a and b chains of the TCR.
Journal of Pharmacology and Experimental Therapeutics, 2020
We hypothesized that in mice histamine via the histamine receptor subtype 4 (H 4 R) on colon epit... more We hypothesized that in mice histamine via the histamine receptor subtype 4 (H 4 R) on colon epithelial cells affects epithelial barrier integrity, perturbing physiological function of the colonic mucosa, and thus aggravating the severity of colitis. In order to test this hypothesis, bone marrow-chimeric mice were generated from H 4 R knockout (H 4 R-/-) and wild-type (WT) BALB/cJ mice and subjected to the DSSinduced acute colitis model. Clinical symptoms and pathohistological derangements were scored. Additionally, total RNA was extracted from either mouse whole-colon homogenates or primary cell preparations enriched for epithelial cells, and gene expression was analyzed by real-time qPCR. The impact of the H 4 R on epithelial barrier function was assessed by measurement of transepithelial electrical resistence of organoid-derived 2D-monolayers from H 4 R-/and WT mice using chopstick electrodes. Bone marrow chimeric mice with genetic depletion of the H 4 R in non-hematopoietic cells exhibited less severe DSS-induced acute colitis symptoms as compared to WT mice, indicating a functional, proinflammatory expression of H 4 R in non-immune cells of the colon. Analysis of H 4 R expression revealed the presence of H 4 R mRNA in colon epithelial cells. This expression could be confirmed and complemented by functional analyses in organoid-derived epithelial cell monolayers. Thus, we conclude that the H 4 R is functionally expressed in mouse colon epithelial cells, potentially modulating mucosal barrier integrity and intestinal inflammatory reactions, as was demonstrated in the DSS-induced colitis model, where presence of the H 4 R on non-hematopoietic cells aggravated the inflammatory phenotype.
Naunyn-schmiedebergs Archives of Pharmacology, Jun 10, 2023
In previous studies, we demonstrated the involvement of H 4 R in inflammatory bowel disease (IBD)... more In previous studies, we demonstrated the involvement of H 4 R in inflammatory bowel disease (IBD) and IBD-associated colon cancer in mice and could ascribe H 4 R-mediated histamine function to colon epithelial cells. The transferability of obtained data to humans is however lacking. Functional expression of H 4 R on colon epithelial cells is a prerequisite to pursue the hypothesis of involvement of H 4 R in carcinogenesis. Thus, we here compared the expression of histamine receptor subtypes in a series of cell lines. Out of these, three colon-derived cell lines displaying different combinations of H 1 R and H 4 R expression were submitted to functional analyses. Human hematopoietic HMC-1, HL-60, and U937, lung-derived A549 and Calu-3, and colorectal LoVo, SW 480, Caco-2, HT-29, and HCT116 cells were included in the study. mRNA expression was quantified by RT-qPCR. For functional analyses, Caco-2, HT-29, and HCT116 cells were treated by incubation with 1-10 µM histamine in the presence or absence of selective histamine receptor antagonists. Calcium mobilization, cAMP accumulation, and cell proliferation were measured by fluorimetry, mass spectrometry, and real-time bioimpedance measurements, respectively. Histamine receptor expression was heterogeneous in the cell lines tested. In most cell lines, we detected H 1 R mRNA while H 4 R mRNAs were found only occasionally. The colon-derived epithelial cell lines LoVo, SW480, and HT-29 expressed H 1 R mRNA exclusively, while in HCT116 cells H 1 R and H 4 R mRNAs and in CaCo-2 H 2 R mRNA were detectable. Subsequent functional analyses in HT29, Caco-2, and HCT116 cells, however, indicated that only HT-29 responded to histamine stimulation, by means of H 1 R. For a detailed analysis of histamine receptor function, esp. that of H 1 R and H 4 R, in human colon-derived cell lines, the cell lines tested here are not fully convenient unless genetically modified.
Three cell surface molecules participate in lnterleukin-1 (IL-1) binding and signal generation, t... more Three cell surface molecules participate in lnterleukin-1 (IL-1) binding and signal generation, the two distinct types of receptors (type ! IL-IR and type II IL-IR) and the IL-1 receptor accessory protein 0L-1RAcP). Low surface expression hampers the detection of all three components on a protein level in most cell types, thus the highly sensitive RT-PCR was used to analyse the mRNA expression in a panel of 18 murine cell types of different hemopoietic lineages and fibroblasts. The transcription of both types of IL-1 receptors was detected in all cell lines tested. In most cell lines the IL-IRAcP was co-expressed with the IL-I receptors, and only these lines responded to IL-I. However, in three cell lines no mRNA for the IL-IRAcP could be detected, and these cells did not respond to [L-I. These results suggest that the expression of the IL-IRAcP correlates with IL-I responsiveness and they point to a pivotal role for the IL-IRAcP in IL-I signal generation.
The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allerg... more The anaphylatoxins C3a and C5a are involved in the pathophysiology of microbial as well as allergic inflammation in the lungs. Besides their expression in leukocytes, receptors for C3a and C5a (C3aR and C5aR) have been noted in alveolar and bronchial epithelial cells, bronchial smooth muscle cells as well as in vascular endothelial and smooth muscle cells of normal and inflamed human and murine lungs. Recently, however, expression of anaphylatoxin receptors in parenchymal cells of the lung (and kidney) has been challenged. Using well-characterized monoclonal antibodies (mabs) against murine and rat anaphylatoxin receptors, we reexamined the pulmonary distribution of C3aR and C5aR. Immunohistochemistry was performed on frozen sections of lung tissues from normal mice and rats as well as from animals subjected to lipopolysaccharide (LPS)-induced inflammation or from MRL/lpr mice suffering from autoimmune disease. Furthermore, ovalbumin (OVA)-induced models of allergic asthma in the rat and mouse were investigated. Prominent expression of both anaphylatoxin receptors was detectable in resident as well as infiltrating leukocytes. No C3aR protein was observed in alveolar macrophages. Upon LPS-and OVA-challenge as well as in autoimmune inflammation, numbers of infiltrating leukocytes expressing prominent amounts of anaphylatoxin receptors increased. Even under these highly inflammatory conditions, however, expression of C3aR and C5aR was not inducible in parenchymal cells. Thus, our findings identify infiltrating leukocytes as a prominent source of anaphylatoxin receptors in inflamed lungs. A direct involvement of parenchymal cells in anaphylatoxin-mediated pulmonary inflammation is unlikely.
Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to f... more Miltenyi Biotec provides products and services worldwide. Visit www.miltenyibiotec.com/local to find your nearest Miltenyi Biotec contact. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. autoMACS, gentleMACS, MACS, and MACSQuant are registered trademarks or trademarks of Miltenyi Biotec GmbH. All other trademarks mentioned in this document are the property of their respective owners and are used for identification purposes only.
Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, a... more Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, as detected by saturation binding and RT-PCR, and are responsive to IL-1beta activation by producing inflammatory cytokines IL-6 and IL-8. IL-1beta can also have an indirect effect on nervous cell functions, since it is able to modulate the stimulus-induced increase of intracellular Ca++ levels, one of the first steps of the cell activation mechanism. In fact, on SK-N-SH neuroblastoma cells, IL-1beta can inhibit the Ca++ increase induced by stimulation of acetylcholine receptors with carbachol. In parallel to IL-1beta, the neurotrophic factor CNTF also shows an inhibitory effect on carbachol-stimulated Ca++ increase in CNTFRalpha-expressing SK-N-SH cells. However, when simultaneously present, the two cytokines cross-inhibit, thus allowing full cell activation in response to the cholinoceptor agonist. The inhibitory effect of CNTF on IL-1beta activities on nervous cells was confirmed in th...
<p>MRL<sup>lpr</sup> mice displaying an IL-18 expression status as indicated we... more <p>MRL<sup>lpr</sup> mice displaying an IL-18 expression status as indicated were housed as described in the methods section. Individual ages (weeks) when they reached a health status ranking score ≥4 (<a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0140173#pone.0140173.t001" target="_blank">table 1</a>) were documented. Presented are pooled data from n = 23, 38, and 51 individuals of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> mice, respectively. The three data sets were analysed by Log-rank (Mantel-Cox) test and revealed no statistically significant differences.</p
<p>HEK293 mH<sub>1</sub>R cells (A+C) and HEK293 mH<sub>4</sub>R ce... more <p>HEK293 mH<sub>1</sub>R cells (A+C) and HEK293 mH<sub>4</sub>R cells (B+D) were incubated in the presence or absence of 50 ng/ml pertussistoxin (PTX) for 18 h. Changes in [Ca<sup>2+</sup>]<sub>i</sub> were determined as described in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g002" target="_blank">Figure 2</a> after stimulation with 100 µM histamine (HA) or 10 µM ATP as indicated on the x-axis (A+B). Intracellular cAMP concentrations were analyzed as described in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0107481#pone-0107481-g003" target="_blank">Figure 3</a> after stimulation for 10 min with 100 µM forskolin (Forsk) and histamine (HA) (C+D). Forskolin-induced cAMP concentrations were 2385±160 pmol/mg protein in HEK293 H<sub>1</sub>R and 2164±436 pmol/mg protein in HEK 293 H<sub>4</sub>R cells, respectively. Data shown are means ± SD (n = 2, each consisting of 2–3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).</p
<p>Kidneys of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/... more <p>Kidneys of MRL<sup>lpr</sup>, MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> mice were removed, processed, and histologically analysed. (A) Presented are representative pictures of PAS-stained kidney sections obtained from MRL<sup>lpr</sup> (+/+), MRL<sup>lpr</sup>Il18<sup>+/tm</sup>, (+/tm), and MRL<sup>lpr</sup>Il18<sup>tm/tm</sup> (tm/tm) mice. (B) Quantitative evaluations of the sections were performed using the scoring system as detailed in <a href="https://www.plosone.org/article/info:doi/10.1371/journal.pone.0140173#pone.0140173.t002" target="_blank">Table 2</a>. Presented are individual data for three evaluated parameters from 5–11 mice per strain and the respective means +/- SD. Differences between the three strains were analysed by ANOVA with Bonferroni’s post test (*, p < 0.05; no indication: no significance).</p
Histamine is a pro-inflammatory mediator with a prominent role in allergic diseases. Antagonists ... more Histamine is a pro-inflammatory mediator with a prominent role in allergic diseases. Antagonists at the histamine receptor subtype 1 are central in anti-allergic therapies, with the exception of allergic asthma, where they are clinically without effect. The latest identified histamine receptor subtype 4, which is expressed mainly in hematopoietic cells, now provides a reasonable target for new therapeutic strategies inhibiting histamine function. The pathophysiology of allergy, esp. allergic asthma, and in its context the effects of antagonists at the histamine receptor subtype 4 in preclinical and clinical settings are discussed in this chapter.
... In genetic immunisation studies it was shown that far less DNA has to be applied by gene-gun ... more ... In genetic immunisation studies it was shown that far less DNA has to be applied by gene-gun as compared to needle injection, to obtain the same level of expression of the exogenous gene (Barry and Johnston, 1997; Feltquate et al, 1997; Pertmer et al, 1995). ...
Idiotype The term idiotype indicates the individuality of a particular BCR or TCR. It consists of... more Idiotype The term idiotype indicates the individuality of a particular BCR or TCR. It consists of a collection of conformational antigenic determinants (idiotopes) which are located within the combined variable domains of antibody H chains and L chains (F V fragment) or the a and b chains of the TCR.
Journal of Pharmacology and Experimental Therapeutics, 2020
We hypothesized that in mice histamine via the histamine receptor subtype 4 (H 4 R) on colon epit... more We hypothesized that in mice histamine via the histamine receptor subtype 4 (H 4 R) on colon epithelial cells affects epithelial barrier integrity, perturbing physiological function of the colonic mucosa, and thus aggravating the severity of colitis. In order to test this hypothesis, bone marrow-chimeric mice were generated from H 4 R knockout (H 4 R-/-) and wild-type (WT) BALB/cJ mice and subjected to the DSSinduced acute colitis model. Clinical symptoms and pathohistological derangements were scored. Additionally, total RNA was extracted from either mouse whole-colon homogenates or primary cell preparations enriched for epithelial cells, and gene expression was analyzed by real-time qPCR. The impact of the H 4 R on epithelial barrier function was assessed by measurement of transepithelial electrical resistence of organoid-derived 2D-monolayers from H 4 R-/and WT mice using chopstick electrodes. Bone marrow chimeric mice with genetic depletion of the H 4 R in non-hematopoietic cells exhibited less severe DSS-induced acute colitis symptoms as compared to WT mice, indicating a functional, proinflammatory expression of H 4 R in non-immune cells of the colon. Analysis of H 4 R expression revealed the presence of H 4 R mRNA in colon epithelial cells. This expression could be confirmed and complemented by functional analyses in organoid-derived epithelial cell monolayers. Thus, we conclude that the H 4 R is functionally expressed in mouse colon epithelial cells, potentially modulating mucosal barrier integrity and intestinal inflammatory reactions, as was demonstrated in the DSS-induced colitis model, where presence of the H 4 R on non-hematopoietic cells aggravated the inflammatory phenotype.
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