Levels Of GRO-alpha Research Articles

Abstract 4669: Circulating cytokines as biomarkers of BEP toxicity in TGCTs patients

Abstract Background: Cytokines are key components in process of inflammation and immune response, what are important processes in many toxicities. Bleomycin, etoposide, cisplatinum (BEP) is a standard treatment in patients with testicular germ cell tumors (TGCTs). Grade 3/4 toxicity can be dose limiting and can highly influence patient’s quality of life. Our aim was to investigate correlation between circulating cytokines and BEP toxicity in TGCTs patients. Methods: The study population consisted of chemonaive TGCTs patients treated by BEP chemotherapy. Grade 3 and 4 toxicities were assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 after first cycle of chemotherapy. G-CSF was administered to all patients after chemotherapy. Circulating 51 plasma cytokines were analyzed at baseline before chemotherapy administration (94 patients) and on cycle 1 day 22 (57 patients) using multiplex bead arrays. Toxicities were correlated to pretreatment and also postreatment levels of circulating cytokines. Spearman's correlation test was used to analyze the association between cytokines and toxicity. Results: Significant correlations between grade 3/4 toxicity and cytokines were observed. Neutropenia was associated with elevated pretreatment levels of TGF-beta 2, IL-2R alpha, HGF, MIG, NGF-beta, SDF-1 alpha, IL-1beta, IL-4, IL-5, IL-17, FGF-basic, G-CSF, MIP-1 alpha and postreatment elevated levels of GRO-alpha, TNF-beta. Thrombocytopenia related with higher pretreatment levels of IL-2R alpha, IL-18, M-CSF and higher postreatment IL-3, IL-12p40, GRO-alpha, MCP-3, M-CSF, MIF, NGF -beta. Anemia correlated with elevated pretreatment IL-16, GRO-alpha, HGF, MIF, NGF-beta, SCF, SCGF-beta, MCP-1-MCAF and elevated postreatment levels of IL-16, HGF, M-CSF, SCGF-beta. There were many positive associations between pyelonephritis and pretreatment IL-2R alpha, IL-18, M-CSF, NGF-beta and postreatment IL-2R alpha, IL-3, IL12p40, CTACK, HGF, MCP-3, M-CSF, NGF-beta, SCGF-beta, IL-6. Diarrhea related with higher pretreatment levels of IL-15 and also higher postreatment IL-15, GM-CSF and lower postreatment levels of IFN-alpha, CTACK. Several other associations were detected between elevated postreatment levels of cytokines and toxicities: nausea and SCGF-beta, febrile neutropenia and GRO-alpha, TNF-beta, SIRS and GRO-alpha, PDGF-BB. All correlations were significant P <0.05. Conclusions: Circulating cytokines are promising and easily detectable biomarker for predicting BEP toxicity in TGCTs patients. This work was supported by the Slovak Research and Development Agency under the contracts No. APVV-0016-11 and APVV-15-0086. Citation Format: Daniela Svetlovska, Viera Miskovska, Dana Cholujova, Paulina Gronesova, Silvia Cingelova, Michal Chovanec, Zuzana Sycova-Mila, Jana Obertova, Patrik Palacka, Vanda Usakova, Katarina Kalavska, Jan Luha, Dalibor Ondrus, Stanislav Spanik, Jozef Mardiak, Michal Mego. Circulating cytokines as biomarkers of BEP toxicity in TGCTs patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2017-4669

Effect of Obesity on Growth‐related Oncogene Factor‐α, Thrombopoietin, and Tissue Inhibitor Metalloproteinase‐1 Serum Levels

We have recently identified several adipokines as oversecreted by omental adipose tissue (AT) of obese subjects: two chemokines (growth-related oncogene factor-alpha (GRO-alpha), macrophage inflammatory protein-1 beta (MIP-1 beta)), a tissue inhibitor of metalloproteinases-1 (TIMP-1), an interleukin-7 (IL-7) and a megakaryocytic growth-factor (thrombopoietin (TPO)). These adipokines are involved in insulin resistance and atherosclerosis. The objectives of this study were to determine whether the circulating levels of these adipokines were increased in obesity and to identify the responsible factors. A cross-sectional study including 32 lean (BMI (kg/m(2)) <25), 15 overweight (BMI: 25-29.9), 11 obese (BMI: 30-39.9), and 17 severely obese (BMI >40) age-matched women was carried out. Serum adipokine levels, insulin sensitivity, and substrate oxidation were measured by ELISA, euglycemic-hyperinsulinemic clamp, and indirect calorimetry, respectively. Circulating levels of GRO-alpha, TPO, and TIMP-1 were higher in obese and/or severely obese women than in lean ones (+30, 55, and 20%, respectively). Serum levels of these adipokines positively correlated with insulinemia or glycemia, and negatively with insulin sensitivity. TIMP-1 also positively correlated with blood pressure, and TPO with triglyceride levels. Multiple regression analysis showed that fat mass per se was an independent determinant of GRO-alpha, TPO, and TIMP-1 levels, suggesting that hypertrophied adipocytes and recruited macrophages in expanded AT mainly contribute to this hyperadipokinemia. Insulinemia, glycemia and resistance of glucose oxidation to insulin were additional predictors for TPO. Circulating GRO-alpha, TPO, and TIMP-1 levels are increased in obesity. This may be partially due to augmented adiposity per se and to hyperinsulinemia/insulin resistance. These high systemic levels may in turn worsen/promote insulin resistance and cardiovascular disease.

Etanercept treatment reduces the serum levels of interleukin-15 and interferon-gamma inducible protein-10 in patients with rheumatoid arthritis

Tumor necrosis factor-alpha (TNF-alpha) has an essential role in the pathogenesis of rheumatoid arthritis (RA) and has been known to induce the production of several inflammatory molecules in vivo. To analyze in vivo the active mechanism of the TNF-alpha blocking agent, etanercept, the serum levels of the cytokine interleukin-15 (IL-15) and the chemokines growth-regulated protein-alpha (Gro-alpha), and interferon-gamma inducible protein-10 (IP-10) in RA patients were measured. Twenty-two patients with RA were administered etanercept once or twice a week for more than 6 months. The clinical and laboratory parameters were measured and serum levels of IL-15, Gro-alpha, and IP-10 were determined using enzyme-linked immunosorbent assay (ELISA) kits at the baseline and at 3 and 6 months after the initial treatment. Additionally, the production of IL-15 and IP-10 by cultured synovial cells stimulated with TNF-alpha from RA patients was determined by ELISA. A significant decrease in serum levels of IL-15 and IP-10 was observed at 3 and 6 months after initial treatment with etanercept, but not in those of Gro-alpha. TNF-alpha induced production of IP-10, but not IL-15 in cultured synovial cells from RA patients. This study demonstrated for the first time the reduction of IP-10 and IL-15 production in RA patients as active mechanisms of etanercept.

Hepatic Expression of CXC Chemokines Predicts Portal Hypertension and Survival in Patients With Alcoholic Hepatitis

Alcoholic hepatitis (AH) is characterized by hepatocellular damage, inflammation, and fibrosis. We performed a prospective study to associate hepatic expression of the CXC subfamily of chemokines with histology findings and prognosis of patients with AH. Liver biopsy samples from 105 patients with AH and 5 normal liver samples (controls) were evaluated for steatosis, inflammation, fibrosis, and cholestasis. Computer-based morphometric analysis assessed the numbers of infiltrating CD3+ T cells and CD15+ cells (neutrophils); terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining was used to quantify apoptosis. Expression of CXC and CC chemokines and selected signaling components were assessed by quantitative reverse-transcription polymerase chain reaction; protein levels of interleukin (IL)-8 and Gro-alpha also were determined by immunohistochemistry. Serum levels of IL-8 and Gro-alpha were measured by enzyme-linked immunosorbent assay. The Cox regression model identified variables associated with mortality. Most patients (75%) had severe AH; their 90-day mortality rate was 21.9%. In AH liver samples, expression of the CXC subfamily members IL-8, Gro-alpha, CXCL5, CXCL6, CXCL10, and platelet factor 4 was up-regulated and compared with controls. The CC chemokine CCL2, but not CCL5, also was up-regulated. Higher expression levels of IL-8, CXCL5, Gro-gamma, and CXCL6 were associated with worse prognosis. Expression of CXC components correlated with neutrophil infiltration and the severity of portal hypertension. In the multivariate analysis, IL-8 protein levels were an independent predictor of 90-day mortality. IL-8 and Gro-alpha serum levels did not correlate with prognosis. Hepatic expression of CXC components correlates with prognosis of patients with AH. Reagents that target CXC chemokines might be developed as therapeutics.

CC and CXC Chemokines Patterns in Psoriasis Determined by Protein Array Method Were Influenced by Goeckerman’s Therapy

Goeckerman's therapy (GT) of psoriasis is based on daily application of pharmacy grade coal tar on affected skin with subsequent exposure to UV light. The aim of this study was to evaluate the influence of Goeckerman's therapy of psoriasis on the levels of proangiogenic chemokines ENA-78 (CXCL5, Epithelial Cell Derived Neutrophil Attractant-78), GRO alpha (CXCL1, Growth-Related Oncogene), IL-8 (CXCL8, Interleukin-8), MCP-1 (CCL2, Monocyte Chemotactic (Chemoattractant) Protein 1) and RANTES (CCL5, Regulated on Activation of Normal T Cell Expressed and Secreted) in peripheral blood of 22 children's patients with psoriasis. 22 otherwise healthy children serve as a control group. The serum levels of chemokines were determined by commercial membrane protein array technique (RayBiotech, USA). Efficacy of Goeckerman's therapy was delineated by PASI score. Disease activity was significantly diminished by Goeckerman's therapy (p < 0.001). Serum levels of GRO alpha and MCP-1 in patients before GT were significantly higher than those measured in healthy blood donors (GRO alpha: p = 0.0128 and MCP-1: p = 0.0003). Serum levels of GRO alpha, MCP-1 and RANTES were significantly diminished by GT (GRO alpha: p = 0.002, MCP-1: p = 0.048 and RANTES: p = 0.0131). Compared to the healthy controls, serum level of MCP-1 remained significantly increased in psoriasis patients after GT (p < 0.0001). In conclusion, we found that the GT of psoriasis influenced the serum levels of proinflammatory and proangiogenic chemokines, especially GRO alpha, MCP-1 and RANTES. It could be the cause for decreased proangiogenic activity which is described after GT of psoriasis.

Dysregulation of stromal derived factor 1/CXCR4 axis in the megakaryocytic lineage in essential thrombocythemia

This study investigated the involvement of chemokines including stromal derived factor 1 (SDF-1), interleukin 8 (IL-8), growth-related oncogene alpha (GRO-alpha) and their receptors, CXCR4, CXCR2 and CXCR1 in essential thrombocythemia (ET), a chronic myeloproliferative disease characterized by megakaryocytic hyperplasia and high platelet count. Fifty-three ET patients were studied. Plasma levels of SDF-1, IL-8 and GRO-alpha, evaluated by enzyme-linked immunosorbent assay, and flow cytometric analysis of CXCR1 and CXCR2 on the platelet membrane, were found to be normal in ET patients. CXCR4 expression on platelet surface as well as platelet CXCR4 mRNA detected by real-time reverse transcription polymerase chain reaction, were decreased. Platelet CXCR4 internalization rate was normal while SDF-1-induced platelet aggregation was delayed, decreased or absent. Immunohistochemical staining revealed that megakaryocytes were also affected. CXCR4 decrease was not observed either in peripheral white blood cells or in circulating CD34(+) precursors. These results show that CXCR4 is decreased in the megakaryocytic lineage in ET, mainly due to a reduced CXCR4 production, and an abnormal platelet response to SDF-1. This report is the first to describe platelet and megakaryocytic CXCR4 deficiency in a human disease and the presence of this abnormality in a megakaryocytic-related illness highlights the important role of SDF-1/CXCR4 axis in platelet development.

The proinflammatory CXC-chemokines GRO-α/CXCL1 and MIG/CXCL9 are concomitantly expressed in ulcerative colitis and decrease during treatment with topical corticosteroids

Ulcerative colitis is characterized by relapsing mucosal inflammation where the lesions include tissue-damaging granulocytes. In addition, T cells and natural killer (NK) cells play important pathophysiologic roles. Chemokines are a large family of peptides that play key roles in the regulation of inflammation. The CXC-chemokines, growth-related oncogene (GRO)-alpha/CXCL1 and interleukin (IL)-8/CXCL8, both recruit neutrophils and possess mitogenic properties, whereas the interferon-dependent CXC-chemokines monokine induced by gamma-interferon (MIG)/CXCL9, interferon-gamma inducible protein of 10 kD/CXCL10, and IFN-inducible T cell alpha chemoattractant/CXCL11 recruit and activate T cells and NK cells. The expression of CXC-chemokines was studied in eight controls and in 11 patients suffering from ulcerative colitis in the distal part of the colon, before and during topical treatment with corticosteroids. Perfusates (obtained before, after 7 days, and after 28 days of treatment) and pinch biopsies (obtained before and after 28 days of treatment) were collected by colonoscopy. The rectal release of GRO-alpha and MIG was determined by enzyme-linked immunosorbent assay (ELISA), and tissue expression of the chemokines was detected in colonic tissue by immunohistochemistry. In perfusates, high levels of GRO-alpha, IL-8, and MIG were detected compared with controls (p=0.02, 0.005, and p=0.03, respectively). During treatment with corticosteroids, both GRO-alpha and MIG decreased. In clinical nonresponders, characterized by sustained inflammation, the levels of GRO-alpha and MIG remained elevated. Both epithelial cells and granulocytes, present in the submucosa, expressed GRO-alpha and MIG as detected by immunohistochemistry. CXC-chemokines are likely to be important in the pathophysiology of ulcerative colitis and may become targets for novel treatment strategies. In addition, GRO-alpha may serve as a marker of disease activity.

Evaluation of bronchoalveolar lavage fluid from ARDS patients with regard to apoptosis

Apoptosis is thought to play an important role in the development of acute respiratory distress syndrome (ARDS). We evaluated the bronchoalveolar lavage (BAL) fluid from ARDS patients focusing on apoptosis. The study enrolled 31 ARDS patients and 20 healthy controls. BAL fluid levels of caspase-cleaved cytokeratin-18 (CK-18) and soluble mediators such as interleukin-8 (IL-8), soluble Fas (sFas), soluble Fas ligand (sFasL), growth-related oncogene-alpha (GRO-alpha), granulocyte colony-stimulating factor (G-CSF), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) were measured using enzyme-linked immunosorbent assay (ELISA). The BAL fluid caspase-cleaved CK-18 levels in ARDS patients were higher than those in controls, reflecting increased epithelial apoptosis, and were correlated with lung injury scores (rs=0.49). The BAL fluid levels of all mediators were significantly higher in ARDS patients than in controls. In ARDS patients, the BAL fluid IL-8 level was positively correlated with the levels of sFas (rs=0.57), GRO-alpha (rs=0.47), and TRAIL (rs=0.45). The BAL fluid IL-8 (rs=0.61), sFas (rs=0.57), G-CSF (rs=0.44), and TRAIL (rs=0.33) levels were correlated with the BAL fluid neutrophil count. The G-CSF levels were significantly higher in non-surviving than in surviving ARDS patients [median 183.4 pg/mL (interquartile range 76.7-315.9) vs. 63.8 pg/mL (36.2-137.2); p<0.05]. The sFas levels were positively correlated with the PaO2/FiO2 ratio (rs=0.40), and the TRAIL levels were negatively correlated with the multiple organ dysfunction scores (rs=-0.37). Among the mediators in BAL fluid from ARDS patients, G-CSF had the most significant prognostic implications, and the sFas and TRAIL levels were correlated with clinical severity.

Cord Blood Cytokine Profile Detection in Neonates with T1D Parents – Monitoring of Cellular Auto‐reactivity Using Protein Microarray

Type 1 diabetes (T1D) is a great medical challenge and its incidence rises rapidly. T lymphocytes and their cytokine production are supposed to play a major role in T1D development. So far, there is no potent tool to recognize the early signs of cellular auto-reactivity which leads to beta-cell damage. The naïve immune system of the newborn (not yet influenced by external factors) can be used as an important model for T1D pathogenesis studies. Cord blood samples of 22 healthy neonates born at term to a diabetic parent (T1DR) and 15 newborns with no family history of any autoimmune disease (controls) were collected. Determination of 23 cytokines was performed before and after the stimulation with diabetogenic autoantigens using protein microarray. We observed lower basal production of all detected cytokines in the T1DR group - granulocyte/macrophage colony-stimulating factor (GM-CSF) (P = 0.025), growth regulated protein (GRO) (P = 0.002), GRO-alpha (P = 0.027), interleukin (IL)-1-alpha (P = 0.051), IL-3 (P = 0.008), IL-7 (P = 0.027), IL-8 (P = 0.042), monocyte chemoattractant proteins (MCP)-3 (P = 0.022), monokine-induced by IFN-gamma (MIG) (P = 0.034) and regulated upon activation normal T-cell express sequence (RANTES) (P = 0.004). Exclusively lower post-stimulative levels of G-CSF (P = 0.030) and GRO-alpha (P = 0.04) were observed in controls in comparison with the basal levels. A significant post-stimulative decrease in G-CSF (P = 0.030) and MCP-2 (P = 0.009) levels was observed in controls in comparison with T1DR neonates. We also observed the interesting impact of the risky genotype on the protein microarray results. Protein microarray seems to be a useful tool to characterize a risk pattern of the immune response for T1D also in newborns.

The Effects of Platelet‐Activating Factor on the Secretion of Interleukin‐8 and Growth‐Regulated Oncogene α in Human Immortalized Granulosa Cell Line (GC1a)

To investigate the role of platelet-activating factor (PAF) in human ovulation, we studied the regulation of interleukin (IL)-8 and growth-regulated oncogene (GRO) alpha in cultured human immortalized granulosa cell line (GC1a). GC1a was cultured in serum-free medium, and incubated with carbamyl-PAF (C-PAF) and/or PAF receptor antagonist (WEB 2086). The supernatants were collected, and IL-8 and GRO alpha were measured by enzyme-linked immunosorbent assay. After treatment with C-PAF, the levels of IL-8 and GROalpha increased in a time-dependent manner. The levels of IL-8 and GROalpha were significantly increased after treatment with C-PAF in a dose-dependent manner. However, the levels of IL-8 and GROalpha were significantly decreased by treatment with C-PAF and with increasing concentrations of WEB 2086. Our data indicated that IL-8 and GROalpha were regulated by C-PAF. The results suggested that PAF may play an important role in human pre-ovulatory processes involving IL-8 and GROalpha production.

Functional Role of the Chemokine Binding Duffy Antigen Receptor Complex (DARC) in Human Inflammation In Vivo.

Background: The Duffy receptor is a promiscuous receptor for chemokines that binds selected members of both the C-X-C and C-C families with high affinity. Duffy antigen may influence plasma levels of proinflammatory cytokines by acting as a “chemokine sink”. Additionally, Duffy knockout mice experienced an exaggerated response to endotoxin in comparison to wild-type mice. The current trial was designed to elucidate the functional role of the Duffy blood group antigen in human inflammation. We hypothesized that “Duffy negative“ volunteers might show an increased inflammatory response to endotoxin (LPS) infusion in terms of cytokine response. The human endotoxemia model is a well established model of systemic inflammation, where a well defined, self-limited inflammatory stimulus permits the elucidation of key players involved in the inflammatory response. We therefore used this model to investigate the functional role of the Duffy Antigen Receptor Complex (DARC) in the inflammatory response after endotoxin challenge.Methods: Thirty-two healthy male volunteers received an intravenous infusion of 2ng/kg endotoxin, 16 Caucasians (Duffy antigen positive on erythrocytes) and 16 subjects of African descent (“Duffy negatives”). Cytokines, chemokines, as well as their receptors were quantified by ELISA, RT-PCR and FACS.Results: Plasma levels of TNF, IL-6, IL-8 and IL-8 mRNA in whole blood increased to a similar extent in both groups after LPS infusion. In contrast, peak MCP-1 levels at 3 hours were roughly 2-fold higher in Duffy positive subjects 16ng/mL as compared to Duffy negative subjects (7ng/mL p<0.001). Similarly, GRO-alpha levels were 2.5-fold higher in Duffy positive subjects at 2 hours after LPS infusion (210pg/mL vs 85pg/mL; p= 0.0001), whereas baseline values showed no difference between both groups. There was no difference in the LPS induced release of MIP-1 beta (which was used as a negative control). Baseline levels of MCP-1 and GRO-alpha in erythrocyte lysates were significantly higher in Duffy positive individuals, whereas IL-8 and MIP-1beta levels were comparable between both groups. Values of MCP-1 and GRO-α, both known to bind to the Duffy antigen, increased extensively in erythrocyte lysates of Duffy positive subjects over time (P=<0.001 vs. time). In contrast, MCP-1 only moderately changed over time in Duffy negative subjects (P<0.03 vs. time), whereas GRO-alpha remained comparable to baseline at all time points (P=0.2 vs. time). As a consequence, MCP-1 and GRO-alpha were several hundred-fold higher in Duffy positive subjects at all time points and reached peak levels at 2–4 hours. Additionally, IL-8, which also binds to erythrocytes via the Duffy antigen, was roughly 7-fold higher in erythrocyte lysates of Duffy positive subjects 2 hours after LPS infusion. As expected, MIP-1beta remained similar at all time points and was comparable between both groups. The expected increase in CD11b, and decrease in CCR-2 and CXCR-1, CXCR-2 occurred without differences between groups.Conclusion: This study characterized for the first time the in vivo function of the Duffy Antigen in humans. The blood group antigen has a profound effect on erythrocyte-bound chemokines and free chemokines in a model of human endotoxemia.

Growth-related oncogene produced in human breast cancer cells and regulated by Syk protein-tyrosine kinase

Syk, a nonreceptor type of protein tyrosine kinase widely expressed in hematopoietic cells, is a candidate suppressor gene in human breast cancer. Reduced expression of Syk protein is correlated with poor prognosis, while its overexpression can reduce the malignant phenotype of breast cancer cells. The mechanism of action of Syk remains unclear. In this study, we utilized low Syk-expressing, highly invasive MDA-MB-231 and high Syk-expressing, less invasive MCF-7 breast cancer cells to investigate the possibility that part of the functional effects of Syk are mediated by cytokines known to play roles in cell migration, invasion or metastasis. Using protein array technology, we determined that MDA-MB-231 cells secrete a number of cytokines known to regulate cellular growth and motility. One such cytokine, growth-related oncogene (GRO), has previously not been described in breast cancer. Of the compounds detected in the culture supernatant of MDA-MB-231, GRO was the only one that was significantly altered by modulation of Syk expression; overexpression of Syk caused a marked reduction in secreted levels of GRO. Conversely, downregulation of the relatively high levels of Syk produced in MCF-7 cells upregulated GRO secretion. At the mRNA level, overexpression of Syk in MDA-MB-231 differentially regulated the 3 GRO isotypes such that message levels of GROalpha and gamma were downregulated while that of GRObeta was not affected. Matrigel invasion assays demonstrated a link between Syk expression and resulting GRO activity in mediating the invasive potential of MDA-MB-231 cells. In summary, our findings provide evidence that human breast cancer cells express and secrete GRO and implicate this cytokine as an essential mediator of the antiinvasive properties of Syk tyrosine kinase.

Possible modulators of IL-8 and GRO-alpha production by granulosa cells.

Interleukin-8 (IL-8) and growth-regulated oncogene-alpha (GRO-alpha) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL-1alpha, tumor necrosis factor-alpha (TNF-alpha), protein kinase C (PKC) activators (TPA), and db-cyclic adenosine monophosphate (cAMP) on IL-8 and GRO-alpha production by immortalized GC1a and granulosa-lutein cells. Confluent granulosa-lutein cells were placed in serum-free medium before incubated for 8 hr with the above-mentioned test agents. Finally, we measured IL-8 and GRO-alpha levels in the culture media using an enzyme-linked immunosorbent assay (ELISA). Treatment of granulosa-lutein cells with of IL-1alpha (1 nM), TNF-alpha (1 nM), TPA (1 nM) and db-cAMP (100 microM) produced higher levels of IL-8 than untreated cells by 8 hr (2274.7 +/- 146.3, 1489.8 +/- 190.1, 1452.9 +/- 152.7, 1313.6 +/- 48.4 pg/mL, respectively; control = 457.7 +/- 38.2 pg/mL; P < 0.001). Treatment of granulosa-lutein cells with 1 nM of IL-1alpha, TNFalpha, TPA, and db-cAMP (100 microM) resulted in higher levels of GRO-alpha than untreated cells by 8 hr (993.7 +/- 9.5, 171.4 +/- 6.5, 147.5 +/- 6.7, 472.4 +/- 16.2 pg/mL respectively; control = 73.8 +/- 8.2 pg/mL; P < 0.001). Our data strongly suggests roles for IL-1alpha, TNFalpha, and PKC activators in the inflammation-like mechanism of human ovulation. Furthermore, our study suggests a positive, but still debatable, role for cAMP in the same mechanism.

Levels of the chemokines growth-related oncogene alpha and epithelial neutrophil-activating protein 78 are raised in patients with severe acute pancreatitis.

Multiple organ dysfunction syndrome secondary to systemic leucocyte activation is the major cause of death following an attack of acute pancreatitis. Although plasma levels of interleukin (IL) 8 are known to be raised in acute pancreatitis, levels of other CXC chemokines such as growth-related oncogene (GRO) alpha and epithelial neutrophil-activating protein (ENA) 78, which are also potent neutrophil chemoattractants and activators, have not been measured. Timed plasma samples were obtained from 51 patients with acute pancreatitis, 27 with a severe attack and 24 with mild disease according to the Atlanta classification. Samples were analysed to determine levels of C-reactive protein (CRP), IL-8, GRO-alpha and ENA-78. Plasma levels of IL-8, GRO-alpha and ENA-78 were increased in patients with severe as opposed to mild acute pancreatitis as early as 24 h following disease onset. Using cut-off levels of 7 pg/ml for IL-8, 70 pg/ml for GRO-alpha and 930 pg/ml for ENA-78, peak levels within the first 24 h of admission had an accuracy of 81, 71 and 87 per cent respectively in predicting the severity of an attack of acute pancreatitis. In patients with severe acute pancreatitis plasma levels of GRO-alpha and ENA-78 were raised in addition to those of IL-8, suggesting that all three chemokines are involved in the inflammatory response in this condition.

Chemokines and interleukin-18 are up-regulated in bronchoalveolar lavage fluid but not in serum of septic surgical ICU patients.

Our objective was to investigate the levels of chemokines (MIP1-alpha, MCP-1, and Gro-alpha), Interleukin-18 (IL-18), and Interleukin (IL-6) in bronchoalveolar lavage (BAL) fluid and serum at the onset and ongoing states of sepsis as defined by the American College of Chest Physicians/Society of Critical Care Medicine in septic surgical ICU patients. Our summary background data was to understand the significance of compartmentalized inflammatory mediator production in an immunologically active organ (lung) in comparison with levels in the systemic circulation. The study group consisted of 20 septic patients and 10 non-septic patients on surgical ICU. At the onset of sepsis, both BAL fluid and serum samples were taken and levels of MIP-1alpha, MCP-1, GRO-alpha, IL-18, and IL-6 were measured by ELISA. Furthermore, over a subsequent 8-day period, levels of these mediators were determined in serum. In some experiments, IL-18 mRNA levels were determined in peripheral blood lymphocytes (PBL) of septic and non-septic patients. At the onset of sepsis, MIP-1alpha, MCP-1, GRO-alpha, IL-18, and IL-6 levels were significantly up-regulated in BAL fluid as compared with non-septic controls. In marked contrast, with the exception of IL-18 mRNA and IL-6 peptide, there was no increase in serum levels of inflammatory mediators determined both at the onset and during the ongoing states of sepsis. Based on the present data, monitoring levels of serum chemokines and IL-18 protein as markers of sepsis might be misleading since despite their non-detection in serum, they were highly up-regulated in the lung tissue compartment. These data might underscore the role of MIP-1alpha, MCP-1, GRO-alpha, and IL-18 in the mediation of local tissue damage. Furthermore, these findings raise the notion that mediator measurement in immunologically active organs might serve as pivotal indicators of sepsis prior to the actual fulfillment of specific clinical criteria that defines the patient as being septic.

Growth Regulated Oncogene-α expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC Receptor-2 dependent mechanism

Growth Regulated Oncogene-alpha (GRO-alpha) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-alpha, designated mGRO-alpha, or KC. We have examined the possible role of mGRO-alpha expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 deficient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-alpha and malignant potential with a pActin-KC vector encoding mGRO-alpha enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in infiltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 deficient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-alpha can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway. Oncogene (2000) 19, 3477 - 3486

Enhanced levels of C-X-C chemokine, human GROalpha, in Helicobacter pylori-associated gastric disease.

C-X-C Chemokines play an important role for neutrophil extravasation through microvessels. Although the level of interleukin (IL)-8 is known to increase in the Helicobacter pylori-infected gastric mucosa, another C-X-C chemokine, GROalpha, has not been evaluated in the H. pylori-associated gastric mucosal injury. The present study was designed to investigate gastric contents of GROalpha in relation to those of IL-8 in the gastric mucosa of H. pylori-infected peptic ulcer patients. Thirty-eight patients with gastric ulcer and 41 with gastritis underwent endoscopy with informed consent and 49 were found to be H. pylori positive and 30 H. pylori negative. Biopsies from the gastric corpus were performed in each patient to examine the H. pylori colonization by bacterial culture, the rapid urease test and histological specimens as well as measurement of the contents of human GROalpha and IL-8. Helicobacter pylori infection was eradicated in 21 patients by triple therapy (lansoprazole 30 mg, amoxycillin 2.0 g, clarithromycin 600 mg; 2 weeks). The samples for GROalpha and IL-8 assay were homogenized in 0.02% aprotinin containing phosphate-buffered solution and the mucosal contents of GROalpha and IL-8 in the supernatants were quantified by sandwich enzyme immunoassay methods. The levels of GROalpha and IL-8 in H. pylori-positive gastric mucosa were significantly higher than those in the H. pylori-negative mucosa. There was a significant linear correlation between the levels of GROalpha and IL-8 (r = 0.798, P < 0.01). After the eradication of H. pylori by the triple therapy, the levels of GROalpha and IL-8 were significantly decreased. The GROalpha showed an increase in the H. pylori-positive gastric mucosa in a similar fashion as IL-8 contents, suggesting a pathogenetic role for GROalpha in H. pylori-associated gastric mucosal injury.

Mucosal chemokine activity in Helicobacter pylori infection.

We examined secretion, mRNA expression, and histologic localization of interleukin-8 (IL-*) and growth-related gene product-alpha (GRO alpha) in the Helicobacter pylori-infected gastric antral mucosa. Antral biopsies were obtained from an area of endoscopically intact mucosa. Significantly higher levels of IL-8 and GRO alpha were secreted in organ cultures from patients with H. pylori infection, and their elevation was prominent in patients with duodenal ulcer. There was a significant association between these alpha-chemokine levels and histologic grades of activity, inflammation, and H. pylori density. In fresh antral biopsies, IL-8 and GRO alpha mRNA expression was detected more frequently in H. pylori-infected patients compared with those without infection. Immunofluorescence microscopy showed localization of IL-8 and GRO alpha proteins in gastric epithelial cells and infiltrating CD68+ macrophages. In the chemotaxis assay, a significant positive correlation was found between neutrophil migration induced by the organ culture supernatants and their contents of IL-8 and GRO alpha. After H. pylori eradication, a significant decrease was observed in IL-8 and GRO alpha levels detected in organ cultures. In conclusion, mucosal alpha-chemokine activity correlates well with histologic severity of H. pylori-associated antral gastritis and can be used to predict the effects of H. pylori eradication therapy.

Chemokine levels in human liver homogenates: Associations between GRO alpha and histopathological evidence of alcoholic hepatitis

Alcoholic hepatitis is characterized by parenchymal neutrophil infiltration. Hepatic synthesis of the neutrophil chemokine interleukin-8 (IL-8) is highly elevated in alcoholic hepatitis and levels correlate with the degree of neutrophil infiltration. The aim of this study was to further determine the spectrum of synthesis of chemokines in liver tissue from patients with alcoholic liver disease and a range of disease control subjects. Subjects were composed of 24 patients with alcoholic liver disease of whom 15 had histopathological evidence of alcoholic hepatitis (10 cirrhotic) and 9 no evidence of alcoholic hepatitis (5 cirrhotic); other controls included; normal liver (n = 6), viral hepatitis (n = 16), primary biliary cirrhosis (n = 5), acute liver failure (n = 4), and miscellaneous liver disease (n = 13). Levels of the C-X-C neutrophil chemokine GRO alpha and the mononuclear cell C-C chemokines: macrophage inflammatory protein 1 alpha, macrophage chemotactic protein 1 and RANTES, were determined by ELISA in liver homogenates. Levels of the neutrophil chemokine GRO alpha were specifically elevated (mean 46 pg/mg, compared with normal liver 11 pg/mg) in patients with alcoholic hepatitis. GRO alpha levels correlated with IL-8 levels and were higher in patients with alcoholic liver disease and parenchymal neutrophil infiltration. Hepatic RANTES was elevated in diseased liver, with the highest levels found in viral hepatitis (mean 117 pg/mg, compared with 24 pg/mg in normal liver). No significant changes in hepatic levels of macrophage inflammatory protein 1 alpha (MIP-1 alpha) or macrophage chemotactic protein 1 (MCP-1) were found. These data provide further supportive evidence that parenchymal neutrophil infiltration in alcoholic hepatitis may be determined by selective upregulation of C-X-C chemokine synthesis. (Hepatology 1996 Nov;24(5):1156-60)