Expression Of ICAM-1 Adhesion Molecules Research Articles

Cold Exposure Induces Vaso-Occlusion and Hyperalgesia in Mice with Sickle Cell Disease That Is Dependent on Complement Activation

BACKROUND AND AIM: Vaso-occlusive crises (VOC) cause severe pain in patients with sickle cell disease (SCD). To date, epidemiological studies have shown seasonably colder temperatures exacerbate SCD-related pain. Although literature and clinical observations suggest a connection between cold and triggering of acute VOC pain, the biologic mechanisms underlying VOC-evoked pain in SCD patients are poorly understood. Repeated cold or painful events results in vasoconstriction and decreased perfusion, which increases red cell transit time, deoxygenation, and hemoglobin S polymerization [Veluswamy et al. Blood. 2020;136:1191]. SCD mice exhibit ischemia-reperfusion (I/R) pathophysiology in response to hypoxia-reoxygenation with a concomitant increase in hyperalgesia, inflammation, and complement activation. Because complement activation plays a crucial role in animal pain models, we used a new model of cold-evoked VOC and I/R in HbSS mice to examine complement C5a/C5aR signaling as a mechanistic link between vaso-occlusion and acute pain in SCD. MATERIALS AND METHODS: We used a dorsal skinfold chamber model to measure microvascular stasis (vaso-occlusion) and von Frey filaments applied to the plantar surface of the hind paws to assess mechanical hyperalgesia (pain) in Townes HbSS and control HbAA mice. Non-hyperalgesic mice were exposed to cold (10oC/50oF) for 1 hour (h) and then returned to room temperature. Measurements of VOC and paw withdrawal threshold were collected at baseline before cold and at 1-4 h after cold exposure. After the 4 h stasis measurement, mice were sacrificed, and livers removed and frozen. Hepatic microsomes and nuclear extracts were isolated by differential centrifugation and liver inflammation markers were assessed on Western blots. Hepatic nuclear extracts were blotted with antibodies to NF-ĸB phospho-p65 and total p65. Hepatic microsomes were blotted with antibodies to VCAM-1 and ICAM-1. Plasma samples were blotted with antibodies to alternative pathway factor B activation fragment Bb and complement anaphylatoxin C5a. To examine the role of complement activation in stasis and hyperalgesia produced by cold-evoked VOC, HbSS mice were infused 30 minutes before cold exposure with an inhibitory monoclonal antibody (mAb) to murine C5 to prevent C5 cleavage or C5aR to prevent C5a binding to C5aR and subsequent C5a/C5aR signaling, or a control mAb (1.2 mg/kg body weight). RESULTS: Cold exposure induced more microvascular stasis (vaso-occlusion), 1, 2, 3, and 4 h after cold exposure in subcutaneous venules of HbSS mice as compared to HbAA mice exposed to cold and HbSS mice left at room temperature (p<0.001). Cold exposure also evoked mechanical hyperalgesia in HbSS mice 1, 2, and 24 h after cold as compared to baseline prior to cold (p<0.01) and cold exposed HbAA mice (p<0.05). Vaso-occlusion and mechanical hyperalgesia were accompanied by an increase in Bb and C5a levels in the plasma of HbSS, but not HbAA mice 4 h after cold exposure (p<0.01). In addition, there was an increase in NF-κB activation and VCAM-1 and ICAM-1 adhesion molecule expression in the livers of HbSS mice after cold as compared to HbAA mice exposed to cold and HbSS mice unexposed to cold (p<0.01). Importantly, pretreatment of HbSS mice before cold exposure with a blocking mAb to C5 or C5aR inhibited vaso-occlusion (Figure 1A), mechanical hyperalgesia (Figure 1B), and complement activation (Figure 1C) as compared to cold-exposed HbSS mice treated with a control mAb. CONCLUSION: This mouse model of cold-evoked VOC provides an opportunity to examine potential mediators between vaso-occlusion and pain in SCD. Our results demonstrate that in response to cold temperatures, complement activation contributes to vaso-occlusion and acute hyperalgesia, suggesting a linkage between complement activation, vaso-occlusion, and pain during cold-induced VOC. These studies provide a rationale for clinical use of complement inhibitors to prevent and treat pain in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Effect of Antioxidants on the Production of MCP-1 Chemokine by EA.hy926 Cells in Response to IL-6.

An elevated level of circulatory interleukin 6 (IL-6) is a biomarker for cytokine storm of various etiologies, including COVID-19, and contributes to poor prognosis. Vascular endothelial cells are one of the main targets of pathological action of IL-6. IL-6 activates the trans-signaling pathway via the formation of the IL-6/sIL-6Ra/gp130 receptor complex and subsequent activation of the JAK/STAT3 signaling pathway and PI3K/AKT and MEK/ERK kinases in some cases. Previously, it was shown by the authors' group and other researchers that reactive oxygen species (ROS), including mitochondrial ROS (mito-ROS), contribute to the induction of IL-6 expression in the endothelium, mainly due to increased activation of the transcription factor NF-kB. We have also shown that the mitochondria-targeted antioxidant SkQ1 (Plastoquinolyl-10(6'-decyltriphenyl)phosphonium) prevented tumor necrosis factor (TNF)-induced cytokine storm and death in mice. In the aortas of these animals, SkQ1 also prevented the increase in the expression of NF-kB-dependent genes, including the cytokine IL-6 and the chemokine MCP-1. In the current work, the hypothesis of mito-ROS involvement in the IL-6-signaling-mediated proinflammatory gene expression in endothelial cells is tested. SkQ1 suppressed the expression and secretion of the MCP-1 chemokine, induced by IL-6 in combination with sIL-6-Ra, but not the expression of ICAM-1 adhesion molecules in EA.hy926 human endothelial cells. Using specific inhibitors, the authors have shown that, in EA.hy926 cells, IL-6-induced expression of MCP-1 and ICAM-1 depends on the signaling protein and transcription activator STAT3 and, in some cases, on JNK, PI3K, and MEK1/2 kinases and is independent of p38 kinase. In this model, IL-6 induced rapid STAT3 activation, while ERK1/2 activation was less pronounced, and there was no IL-6 effect on Akt and JNK activation. SkQ1 partially suppressed STAT3 and ERK1/2 activation. Thus, we have shown that SkQ1 suppresses not only NF-kB-dependent expression of IL-6 and other proinflammatory genes but also IL-6-induced activation of JAK/STAT3 and STAT3-dependent expression of MCP-1, which probably contributes to the overall therapeutic effect of SkQ1.

The Recovery of Immune System Parameters in Children Following Lymphoblastic Leukemia Therapy – Preliminary Report

Acute lymphoblastic leukemia (ALL) is the most common pediatric neoplasm. Long-term survival is achieved in approximately 80% of patients. One of the more common complications of ALL treatment is immunosuppression. The aim of the study is to assess the reconstruction rate of the most important immune system parameters in children after ALL treatment. The study included 47 children (22 boys, 25 girls) diagnosed and treated for ALL in Department of Pediatric Hematology and Oncology at Wroclaw Medical University. The study used peripheral blood collected at the time treatment was completed and in the first, second, third and sixth months following treatment, then at yearly intervals up to 10 years after treatment. In order to determine the immune status of the tested samples the following parameters were assessed: white blood cell count, absolute lymphocyte count, the proportions of individual subpopulations of lymphocytes - T (CD3 +), Th (CD4 +), Ts (CD8 +), B (CD19 +), NK (CD16 + 56 +), the concentration of immunoglobulins A, G and M, interleukin 10 activity, as well as the expression of ICAM-1 adhesion molecules. At the end of anti-neoplastic therapy a reduction in both the absolute number leukocytes and various subpopulations of lymphocytes was observed. The lower limits of normal range were achieved about two years after the end of treatment. The concentrations of immunoglobulin IgA, IgG and IgM at the end of treatment were within low normal limits. Normal concentrations of immunoglobulin levels and stability were observed about two years after the end of treatment. There was a slow, steady increase in the production of interleukin-10 and the expression of ICAM-1 adhesion molecules. These results confirm previous observations that after ALL treatment children are in an immunocompromised state for up to 12 months, in terms of both humoral and cellular immunity. Knowing the average growth trends for the main immune system parameters after ALL treatment can be important in clinical practice for children in whom immune reconstruction proceeds slowly. Predicting the expected time required to restore immune function could be of help, for example in combating infections and planning vaccinations.

Prostaglandin E 1 encapsulated into lipid nanoparticles improves its anti-inflammatory effect with low side-effect

Prostaglandin E 1 (PGE1) shows various pharmacological activities including anti-inflammation. However, the rapid metabolization and inactivation of the intravenously administered PGE1 during the first passage through the lungs result in significant non-compliance in clinical trials which greatly limits its application. The aim of this work was to prepare the lipid nanoparticles loading PGE1 to improve its anti-inflammatory effect with low side-effect. The experimental results showed that PGE1 loaded lipid nanoparticles (PLNs) could be successfully prepared by high pressure homogenization with particle size 68.1 ± 4.7 nm, zeta potential −3.32 ± 0.37 mV and entrapment efficiency 92.1 ± 1.3%. PLNs exhibited a sustained release with low burst drug release. PLNs could improve the inhibition effects of PGE1 on lipopolysaccharides (LPS)-induced TNF-α expression on macrophage RAW264.7 cells, and improve the inhibition of lymphocyte to endothelial cell adhesion and ICAM-1 adhesion molecule expression on HUVEC and MDA-MB-468 cell membrane. No allergenicity, vascular and muscle irritation were induced in animals by PLNs even at double of the highest drug concentration of clinical infusion. As a result, PLNs could be a more potential delivery system for PGE1 in the treatment of inflammation-related diseases.

VEGF-A stimulation of leukocyte adhesion to colonic microvascular endothelium: implications for inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by increased leukocyte recruitment and subsequent tissue damage. An increase in the density of the microvasculature of the colon during IBD has been suggested, leading to the concept that angiogenesis may play a pathological role in IBD. Increased tissue and serum levels of the angiogenic cytokine VEGF-A have been reported in cases of active IBD. In this study, we examined the hypothesis that VEGF-A exerts a proinflammatory effect on colon microvascular endothelium that contributes to colonic inflammation. Leukocyte adhesion to VEGF-A-stimulated colon microvascular endothelial cells was examined using a parallel-plate hydrodynamic flow chamber. ICAM-1 adhesion molecule expression on colonic microvascular endothelium also was determined in response to VEGF-A stimulation, along with characterization of leukocyte adhesion molecule expression. High-dose VEGF-A (50 ng/ml) stimulation increased neutrophil and T cell adhesion to and decreased rolling velocities on activated endothelium, whereas low-dose VEGF-A (10 ng/ml) was without effect. Colonic endothelium constitutively expressed ICAM-1, which was significantly increased by treatment with 50 ng/ml VEGF-A or 10 ng/ml TNF-alpha but not 10 ng/ml VEGF-A. T cells expressed CD18 and CD11a with no expression of CD11b, whereas neutrophils expressed CD18, CD11a, and CD11b. Finally, VEGF-A-dependent leukocyte adhesion was found to occur in a CD18-dependent manner. These results demonstrate that VEGF-A levels found in IBD exert a proinflammatory effect similar to other inflammatory agents and suggest that this cytokine may serve as an intermediary between angiogenic stimulation and cell-mediated immune responses.