Granzyme B is elevated in esophageal biopsies from children with eosinophilic esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is an immune-mediated antigen-triggered inflammatory disease of the esophagus. Our aim was to investigate inflammatory responses by an ex vivo biopsy provocation-based method, stimulating biopsies with milk, wheat, and egg extracts. METHODS: An experimental study was conducted on esophageal biopsies from children who underwent esophagogastroduodenoscopy. Supernatants were collected before and after stimulation of the biopsies with food extracts and analyzed for 45 different inflammatory markers. Biopsies were also stained for histological analyzes. RESULTS: Study subjects included 13 controls, 9 active EoE, and 4 EoE in remission, median age 12 years. Of the 45 markers analyzed, three had significant differences between controls and patients with active EoE, Granzyme B, (GzmB), IL-1ra, and CXCL8 (p < .05). Levels of GzmB were higher, and levels of IL-1ra were lower in patients with active EoE compared with controls and EoE in remission both at baseline and after food extract stimulation. CXCL8 increased in active EoE compared with controls only after stimulation. The number of histologically detected GzmB-positive cells were significantly higher in patients with active EoE in contrast to control and EoE remission (p < .05). CONCLUSIONS: The levels of the barrier-damaging protease GzmB were higher in the supernatant both before and after stimulation with food extract ex vivo in patients with active EoE. GzmB was also observed histologically in biopsies from patients with active EoE. The presence of elevated serine protease GzmB in esophageal mucosa of children with active EoE suggests a role in the pathogenesis of this disorder.

Associations between Single Nucleotide Polymorphisms from the Genes of Chemokines and the CXCR2 Chemokine Receptor and an Increased Risk of Endometrial Cancer.

Significant relationships with endometrial cancer were demonstrated, both for CCL2, CCL5, and CXCL8 chemokines and for the chemokine receptor CXCR2. The reported case-control study of genetic associations was designed to establish the role of selected single nucleotide polymorphisms (SNPs) of the CCL2, CCL5, CXCL8, and CXCR2 genes in the onset and progression of endometrial cancer. This study was conducted on 282 women, including 132 (46.8%) patients with endometrial cancer and 150 (53.2%) non-cancerous controls. The genotypes for CCL2 rs4586, CCL5 rs2107538 and rs2280789, CXCL8 rs2227532 and -738 T>A, and CXCR2 rs1126580 were determined, using PCR-RFLP assays. The AA homozygotes in CCL5 rs2107538 were associated with more than a quadruple risk of endometrial cancer (p ≤ 0.050). The GA heterozygotes in the CXCR2 SNP were associated with approximately threefold higher cancer risk (p ≤ 0.001). That association also remained significant after certain adjustments, carried out for age, diabetes mellitus, arterial hypertension, or endometrial thickness above 5 mm (p ≤ 0.050). The A-A haplotypes for the CCL5 polymorphisms and T-A-A haplotypes for the CCL2 and CCL5 SNPs were associated with about a twofold risk of endometrial cancer (p ≤ 0.050). In conclusion, CCL2 rs4586, CCL5 rs2107538 and rs2280789, and CXCR2 rs1126580 demonstrated significant associations with an increased risk of endometrial cancer.

Bothrops moojeni venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Interferon Gamma Inhibits CXCL8-Induced Proliferation and Migration of Pancreatic Cancer BxPC-3 Cell Line via a RhoGDI2/Rac1/NF-κB Signaling Pathway.

Interferon gamma (IFN-γ) is a dimeric soluble cytokine and the only type II interferon. Accumulated evidence suggests that IFN-γ inhibits tumor progression. This study investigated the effects of IFN-γ on the proliferation and migration of pancreatic cancer (PC) cells and the underlying mechanism. IFN-γ treatment decreased the expression and secretion of CXCL8 in BxPC-3 PC cells, suppressed the proliferation and migration of these cells, and enhanced their apoptosis, as determined by increased levels of cleaved Caspase-8 and Bax together with reduced expression of Bcl-2. These effects were abolished by overexpression of CXCL8. Moreover, IFN-γ treatment downregulated RhoGDI2 expression. Depletion of RhoGDI2 and Rac1 by using small interfering RNAs and inhibition of NF-κB by BMS-345541 (an IκB kinase [IKK] inhibitor) suppressed expression of CXCL8. Our results indicate that IFN-γ inhibits the proliferation and migration of PC cells by suppressing CXCL8 expression via a RhoGDI2/Rac1/NF-κB signaling pathway.

The role of the IL-8 signaling pathway in the infiltration of granulocytes into the livers of patients with alcoholic hepatitis.

BACKGROUND AND AIM: IL-8 (C-X-L motif chemokine ligase 8) and CXCR2 (C-X-C-motif chemokine receptor 2) are up regulated in alcoholic hepatitis (AH) liver biopsies. One of the consequences is the attraction and chemotactic neutrophilic infiltrate seen at the AH stage of alcoholic liver disease. MATERIALS AND METHODS: Human formalin-fixed, paraffin-embedded (FFPE) liver biopsies from patients who have AH were studied by (2.1) RNA sequencing, (2.2) PCR and (2.3) semi quantitation of specific proteins in biopsy sections using immunohistochemical measurements of antibody fluorescent intensity with morphometric technology. RESULTS: Immunohistochemistry of IL-8 showed that the expression was increased in the cytoplasm of the hepatocytes in AH liver biopsies compared to the controls. IL-8 and ubiquitin were co-localized in the MDBs. Numerous neutrophils were found throughout and satellitosis of neutrophils around MDBs was present. This suggested that IL-8 may be involved in MDB pathogenesis. RNA seq analysis revealed activation by IL-8 which included neutrophil chemotaxis by LIM domain kinase 2 (LIMK2) (17.5 fold increase) and G protein subunit alpha 15 (GNA15) (27.8 fold increase). CONCLUSIONS: The formation of MDBs by liver cells showed colocalization of ubiquitin and IL-8 in the MDBs. This suggested that IL-8 in these hepatocytes attracted the neutrophils to form satellitosis. This correlated with up regulation of the proteins downstream from the IL-8 pathways including LIMK2, GNG2 (guanine nucleotide binding proteins) and PIK3CB (phosphatidyl isitol-4, 5-biophosphate-3-kinase, catalytic subunit beta).

The immune network in thyroid cancer.

The immune system plays critical roles in tumor prevention, but also in its initiation and progression. Tumors are subjected to immunosurveillance, but cancer cells generate an immunosuppressive microenvironment that favors their escape from immune-mediated elimination. During chronic inflammation, immune cells can contribute to the formation and progression of tumors by producing mitogenic, prosurvival, proangiogenic and lymphangiogenic factors. Thyroid cancer is the most frequent type of endocrine neoplasia and is the most rapidly increasing cancer in the US. In this review, we discuss recent findings on how different immune cells and mediators can contribute to thyroid cancer development and progression.

Transcriptional and posttranscriptional regulation of CXCL8/IL-8 gene expression induced by connective tissue growth factor.

Connective tissue growth factor (CTGF), a CCN family member, is a secreted protein regulating cellular functions, including fibrosis, apoptosis, adhesion, migration, differentiation, proliferation, angiogenesis, and chondrogenesis. CTGF increases proinflammatory factor production; however, inflammatory cytokine regulation by CTGF is poorly understood. The aim of this study was to identify novel biological functions and elucidate the functional mechanisms of CTGF. Specifically, the study focused on the ability of CTGF-primed monocytes to secrete interleukin 8 (CXCL8/IL-8) and determined the signaling pathways involved in CTGF-induced CXCL8/IL-8 gene regulation during inflammation. We transfected wild-type or mutant CXCL8/IL-8 promoter-derived luciferase reporter constructs into 293T cells to examine the effect of CTGF on the CXCL8/IL-8 promoter. The results showed that the activator protein-1 and nuclear factor κB binding sites of the CXCL8/IL-8 promoter are essential for CTGF-induced CXCL8/IL-8 transcription. Moreover, the CTGF-induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase, and extracellular signal-regulated kinase (ERK) is involved in this process. In addition, adenosine-uridine-rich elements (AREs) of the CXCL8/IL-8 3'-untranslated region (3'-UTR) reduce CXCL8/IL-8 mRNA stability. To investigate whether CTGF regulates CXCL8/IL-8 gene expression at the posttranscriptional level, we transfected 293 cells with serial luciferase constructs containing different segments of the CXCL8/IL-8 3'-UTR and then stimulated the cells with CTGF. The results suggested that CTGF stabilized luciferase mRNA and increased luciferase activity by regulating the CXCL8/IL-8 3'-UTR. Moreover, the p38 MAPK pathway may contribute to CTGF-induced CXCL8/IL-8 mRNA stabilization.

Cytokine regulation by epidermal growth factor receptor inhibitors and epidermal growth factor receptor inhibitor associated skin toxicity in cancer patients.

AIM: Epidermal growth factor receptor inhibitor (EGFRI) induced skin toxicity has a prognostic value suggesting skin toxicity can be a useful surrogate marker for successful epidermal growth factor receptor (EGFR) inhibition, improved response and survival. But the pathophysiology of EGFRI induced skin toxicity remains elusive. However the involvement of immunological mechanisms has been speculated. This study investigates the possible underlying mechanism of EGFR inhibition associated cytokine production in keratinocytes as well as in patients after treatment with epidermal growth factor receptor inhibitors (EGFRIs). METHODS: Normal human epidermal keratinocytes (NHEK) were incubated for 2 and 24h with different concentrations of EGFRI (erlotinib) for Western blot analysis and cytokine expression analysis, respectively. Inhibition of EGFR, extracellular-signal-regulated kinase 1/2 (Erk 1/2) and c-Jun was examined by Western blot analysis. Cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the NHEK cell supernatant and also in the serum of 186 cancer patients who are followed up for EGFRI induced skin rash. RESULTS: A significant inhibitory effect of EGFRI was seen on EGFR (Y845), Erk 1/2 and c-Jun in a dose dependent manner. Further downstream, increased CC-chemokine ligand 2 (CCL2), CC-chemokine ligand 5 (CCL5) and decreased interleukin-8 (IL-8) or CXCL8 expression was observed in keratinocytes. In EGFRI treated patients, low levels of serum CXCL8 corresponding to stronger EGFR inhibition were associated with a higher grade of skin toxicity (p=0.0016) and a prolonged overall survival (p=0.018). CONCLUSIONS: The results obtained in this study indicate that EGFRI can regulate cytokines by modulating EGFR signalling pathway in keratinocytes. Moreover, serum levels of CXCL8 in EGFRI treated patients may be important for individual EGFRI induced skin toxicity and patient's survival.

Differential gene expression of chemokines in KRAS and BRAF mutated colorectal cell lines: role of cytokines.

AIM: To study KRAS/BRAF mutations in colorectal-cancer (CRC) that influences the efficacy of treatment. To develop strategies for overcoming combination of treatment. METHODS: Five colonic cell-lines were investigated: DLD-1 with KRAS (G13D) mutation, HT 29 and Colo 205 with BRAF (V600E) mutation as well as the wild type (Wt) cell-lines Caco2 and Colo-320. DLD-1 (KRAS), HT-29 (BRAF) and Caco2 (Wt) cell lines were treated with cytokines (TNFα 50 ng, IL-1ß 1 ng and IFNγ 50 ng) and harvested at different time points (1-24 h). KRAS inhibition was performed by the siRNA-approach. Two colorectal cancer cells DLD-1 and Caco2 were used for KRAS inhibition. About 70% confluency were confirmed before transfection with small interferring RNA (siRNA) oligonucleotides. All the synthetic siRNA sequences were designed in our laboratory. Total RNA and protein was isolated from the cells for RT-PCR and Western blotting. Densitometry of the Western blotting was analyzed with the Image J software (NIH). Results are shown as mean ± SD. RESULTS: RT-PCR analysis in non-stimulated cells showed a low basal expression of TNFα and IL-1ß in the DLD-1 KRAS-mutated cell-line, compared to Caco2 wild type. No detection was found for IL-6 and IFNγ in any of the studied cell lines. In contrast, pro-angiogenic chemokines (CXCL1, CXCL8) showed a high constitutive expression in the mutated cell-lines DLD-1 (KRAS), HT-29 and Colo205 (BRAF), compared to wild type (Caco2). The anti-angiogenic chemokine (CXCL10) showed a high basal expression in wild-type, compared to mutated cell-lines. KRAS down-regulation by siRNA showed a significant decrease in CXCL1 and CXCL10 gene expression in the DLD-1 (KRAS) cell-line in comparison to wild type (Caco2) at 72 h after KRAS silencing. In contrast, the specific KRAS inhibition resulted in an up-regulation of CXCL1 and CXCL10. The results of our study show a higher expression of pro-angiogenic chemokines at basal level in mutated cell-lines, which was further increased by cytokine treatment. CONCLUSION: To summarize, basal chemokine gene expression for pro-angiogenic chemokines was high in mutated as compared to wild type cell-lines. This reflects the likely existence of a different microenvironment in tumours consistent of wild type or mutated cells. This may help to rationalize the choice of molecular targets for suitable therapeutic investigation in clinical studies.

A novel phenylcyclohex-1-enecarbothioamide derivative inhibits CXCL8-mediated chemotaxis through selective regulation of CXCR2-mediated signalling.

BACKGROUND AND PURPOSE: Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function. EXPERIMENTAL APPROACH: We used the CXCR2 Tango(TM) assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338. KEY RESULTS: CX4338 selectively inhibited CXCR2-mediated recruitment of ß-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice. CONCLUSIONS AND IMPLICATIONS: A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated ß-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.