Neutralization of IL-8 prevents the induction of dermatologic adverse events associated with the inhibition of epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) inhibitors are widely used in the treatment of cancer. EGFR-targeted treatment is known to be associated with a high incidence of dermatological adverse reactions, including papulopustular rash, which can be dose-limiting and may affect compliance to treatment. Currently, the pathways involved in EGFR inhibitor-induced rash are poorly understood and few treatment options for this adverse event are available. Here, we developed a model for induction of papulopustular rash in healthy human volunteers by subcutaneous injection of the anti-EGFR monoclonal antibody zalutumumab. The injection sites and surrounding skin were evaluated by a dermatologist for the presence or absence of papulopustular rash and skin biopsies were taken to confirm the macroscopical findings by immunohistochemistry. Locally injected zalutumumab induced a papulopustular rash, characterized by acute follicular neutrophil-rich hair follicle inflammation, and thus mimicked adverse events induced by systemic administration of EGFR inhibitors. In this model, we tested the hypothesis that neutrophils, attracted by IL-8, play a central role in the observed rash. Indeed, concomitant local repeat dose treatment with HuMab-10F8, a neutralizing human antibody against IL-8, reduced the rash. Inhibition of IL-8 can therefore ameliorate dermatological adverse events induced by treatment with EGFR inhibitors.     

Baclofen and Adenosine Inhibition of Synaptic Transmission at CA3-CA1 Synapses Display Differential Sensitivity to K<Superscript>+</Superscript> Channel Blockade

The metabotropic GABA_B and adenosine A₁ receptors mediate presynaptic inhibition through regulation of voltage-dependent Ca²⁺ channels, whereas K⁺ channel regulation is believed to have no role at the CA3-CA1 synapse. We show here that the inhibitory effect of baclofen (20 μM) and adenosine (300 μM) on field EPSPs are differentially sensitive to Cs⁺ (3.5 mM) and Ba²⁺ (200 μM), but not 4-aminopyridine (100 μM). Barium had no effect on paired-pulse facilitation (PPF) in itself, but gave significant reduction (14 ± 5%) when applied in the presence of baclofen, but not adenosine, suggesting that the effect is presynaptic and selective on the GABA_B receptor-mediated response. The effect of Ba²⁺ on PPF was not mimicked by tertiapin (30 nM), indicating that the underlying mechanism does not involve GIRK channels. Barium did not affect PPF in slices from young rats (P7–P8), suggesting developmental regulation. The above effects of Ba²⁺ on adult tissue were reproduced when measuring evoked whole-cell EPSCs from CA1 pyramidal neurons: PPF was reduced by 22 ± 3% in the presence of baclofen and unaltered in adenosine. In contrast, Ba²⁺ caused no significant change in frequency or amplitude of miniature EPSCs. The Ba²⁺-induced reduction of PPF was antagonized by LY341495, suggesting metabotropic glutamate receptor involvement. We propose that these novel effects of Ba²⁺ and Cs⁺ are exerted through blockade of inwardly rectifying K⁺ channels in glial cells, which are functionally interacting with the GABA_B receptor-dependent glutamate release that generates heterosynaptic depression.     

The role of adrenaline as a modulator of cardiac performance in two Antarctic fishes

The present work was performed to test the hypothesis that Antarctic teleosts rely mostly on cholinergic inhibition for autonomic modulation of the heart. The effects of adrenaline on the inotropic properties on paced, isometrically contracting muscle strips were examined in two distinct Antarctic teleosts, the haemoglobinless icefish Chaenocephalus aceratus and the red-blooded Notothenia coriiceps. All tissues examined revealed a negative force-frequency relationship. Under baseline conditions C. aceratus contracted with a force twice as great as that of N. coriiceps. While the degree to which ventricular tissues responded to adrenaline varied between species, adrenergic stimulation significantly increases myocyte contraction force in this group of fishes. Contraction and relaxation times were not significantly affected by adrenaline concentration while absolute rates of contraction were. Adrenergic stimulation does not enable tissues to achieve higher contraction frequencies, but is shown to be a potent modulator of contraction force.     

Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands

Renin and epidermal growth factor (EGF) are synthesized in large amounts by the male mouse submandibular glands. We report the peptides to be secreted mainly in an exocrine manner. The highest values in saliva are obtained upon stimulation with the alpha-adrenergic agonist phenylephrine. The median value for renin is 54 700 nmol/l and the median value for EGF is 211 800 nmol/l. Aggressive behaviour and beta-adrenergic stimulation also increase salivary output of both peptides, while vasoactive intestinal polypeptide (VIP) plus pilocarpine selectively stimulate the secretion of renin. The pattern of increase in plasma is comparable to that in saliva though the substance concentration is lower by a factor of 2 to 70 for renin and a factor of 280 to 12 000 for EGF.     

Molecular regulation of MHC class I chain-related protein A expression after HDAC-inhibitor treatment of Jurkat T cells

In this study, we characterize the molecular signal pathways that lead to MHC class I chain-related protein A (MICA) expression after histone deacetylase (HDAC)-inhibitor (HDAC-i) treatment of Jurkat T cells. Chelating calcium with BAPTA-AM or EGTA potently inhibited HDAC- and CMV-mediated MICA/B expression. It was further observed that endoplasmic reticulum calcium stores were depleted after HDAC treatment. NF-kappaB activity can be induced by HDAC treatment. However, nuclear translocation of NF-kappaB p65 was not observed after HDAC treatment of Jurkat T cells and even though we could effectively inhibit p65 expression by siRNA, it did not modify MICA/B expression. To identify important elements in MICA regulation, we made a promoter construct consisting of approximately 3 kb of the proximal MICA promoter in front of GFP. Deletion analysis showed that a germinal center-box containing a putative Sp1 site from position -113 to -93 relative to the mRNA start site was important for HDAC and CMV-induced promoter activity. Sp1 was subsequently shown to be important, as targeted mutation of the Sp1 binding sequence or siRNA mediated down modulation of Sp1-inhibited MICA promoter activity and surface-expression.     

CXCR3 expression on CD34(+) hemopoietic progenitors induced by granulocyte-macrophage colony-stimulating factor: II. Signaling pathways involved

CXCR3, known to have four ligands (IFN-gamma inducible protein 10 (gamma IP-10), monokine induced by IFN-gamma (Mig), I-TAC, and 6Ckine), is predominantly expressed on memory/activated T lymphocytes. We recently reported that GM-CSF induces CXCR3 expression on CD34(+) hemopoietic progenitors, in which gamma IP-10 and Mig induce chemotaxis and adhesion. Here we further report that stimulation with GM-CSF causes phosphorylation of Syk protein kinase, but neither Casitas B-lineage lymphoma (Cbl) nor Cbl-b in CD34(+) hemopoietic progenitors can be blocked by anti-CD116 mAb. Specific Syk blocking generated by PNA antisense completely inhibits GM-CSF-induced CXCR3 expression in CD34(+) progenitors at both mRNA and protein as well as at functional levels (chemotaxis and adhesion). Cbl and Cbl-b blocking have no such effects. Thus, GM-CSF binds to its receptor CD116, and consequently activates Syk phosphorylation, which leads to induce CXCR3 expression. gamma IP-10 and Mig can induce Syk, Cbl, and Cbl-b phosphorylation in CD34(+) progenitors by means of CXCR3. gamma IP-10 or Mig has induced neither chemotaxis nor adhesion in GM-CSF-stimulated Cbl-b-blocked CD34(+) hemopoietic progenitors, whereas SDF-1alpha induces both chemotaxis and adhesion in these cells. Interestingly, gamma IP-10 and Mig can induce chemotaxis and adhesion in GM-CSF-stimulated Syk- or Cbl-blocked CD34(+) hemopoietic progenitors. Thus, Cbl-b, but not Syk and Cbl phosphorylation, is essential for gamma IP-10- and Mig-induced chemotaxis and adhesion in CD34(+) hemopoietic progenitors. This study provides a useful insight into novel signaling transduction pathways of the functions of CXCR3/gamma IP-10 and Mig, which may be especially important in the cytokine/chemokine environment for mobilization, homing, and recruitment during proliferation, differentiation, and maturation of hemopoietic progenitor cells.     

CXCR3 expression and activation of eosinophils: role of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma

CXC chemokine receptor 3 (CXCR3), predominately expressed on memory/activated T lymphocytes, is a receptor for both IFN-gamma-inducible protein-10 (gamma IP-10) and monokine induced by IFN-gamma (Mig). We report a novel finding that CXCR3 is also expressed on eosinophils. gamma IP-10 and Mig induce eosinophil chemotaxis via CXCR3, as documented by the fact that anti-CXCR3 mAb blocks gamma IP-10- and Mig-induced eosinophil chemotaxis. gamma IP-10- and Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA expressions in eosinophils are up- and down-regulated by IL-2 and IL-10, respectively, as detected using flow cytometry, immunocytochemical assay, and a real-time quantitative RT-PCR technique. gamma IP-10 and Mig act eosinophils to induce chemotaxis via the cAMP-dependent protein kinase A signaling pathways. The fact that gamma IP-10 and Mig induce an increase in intracellular calcium in eosinophils confirms that CXCR3 exists on eosinophils. Besides induction to chemotaxis, gamma IP-10 and Mig also activate eosinophils to eosinophil cationic protein release. These results indicate that CXCR3-gamma IP-10 and -Mig receptor-ligand pairs as well as the effects of IL-2 and IL-10 on them may be especially important in the cytokine/chemokine environment for the pathophysiologic events of allergic inflammation, including initiation, progression, and termination in the processes.     

Chromosome-damaging activity of a ruthenium radiosensitizer, RuCl2 (DMSO)2 (4-nitroimidazole)2, in Chinese hamster ovary cells in vitro

The metal complex, RuCl2 (DMSO)2 (4-nitroimidazole)2, 1, which has hypoxic radiosensitizing properties, was examined for genotoxic activity, as measured by the in vitro induction of chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells. A dose-dependent increase in the frequencies of metaphases with chromatid aberrations was observed for 1. Addition of S9 liver microsomal mixture and 1 to the cultured CHO cells did not alter the clastogenic activity noted for the complex itself. The clastogenic (chromosome damaging) activity of a precursor complex, cis-RuCl2(DMSO)4 and the ligand, 4-nitroimidazole (4-NO2-Im) were found to be less than that of 1 at corresponding concentrations. A comparison with two drugs used clinically with radiation, cis-dichlorodiammineplatinum(II) (cis-DDP) and misonidazole (miso), indicated that the clastogenic activity of 1 was similar to miso and much less than that of cis-DDP.     

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.