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The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Pneumonia associated with aspiration of bacterial-laden gastric contents is characterized by Glu-Leu-Arg (ELR)-CXC chemokine (e.g., CXC2L1, CXCL8) expression leading to local neutrophil sequestration. This neutrophil response is designed to be protective, but overly aggressive responses can be pathogenic in themselves. Herein we assessed whether blocking neutrophil responses in a guinea pig model of aspiration pneumonia would foster airway bacterial growth. Guinea pigs (n=5) were challenged intranasally with saline, acidified saline or acidified gastric contents (35mg/kg body weight, pH 2.0) and treated subcutaneously with 250mug/kg of the human ELR-CXC chemokine antagonist CXCL8((3-72))K11R/G31P (G31P) or saline. After 20h the animals' airway inflammatory responses and bacterial burdens were assessed. A loss of vascular integrity was apparent in the lungs of the saline-treated aspiration pneumonia animals (12.07+/-1.3% of the pleural surfaces exhibited hemorrhagic consolidation, 4.6x10(6) RBC/ml bronchoalveolar lavage fluid [BALF]), as was a pulmonary neutrophilia. The BAL fluids contained gram-negative and -positive bacteria (total load, 6.3+/-3.2x10(7) CFU/ml BALF) that are associated with nosocomial infections in humans. The G31P-treatments attenuated the pulmonary vascular complications (2.27+/-0.5% pleural surface hemorrhagic consolidation, 0.46x10(6) RBC/ml BALF), and reduced the pulmonary neutrophilia by >/=86%. The G31P-treatments did not lead to significant changes in the airway bacterial loads (total load, 3.46+/-1.8x10(7) CFU/ml BALF). This data indicates that attenuation of the pulmonary neutrophil response in aspiration pneumonia reduces pathology very significantly but does not reduce the efficiency of pulmonary bacterial clearance., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Background: Cyclooxygenase-2 (COX-2) overexpression by a primary tumor correlates with poor prognosis in breast cancer, including early spread to bone. Interleukin-8 (IL-8) stimulates osteoclastogenesis and resorption of bone, and elevated IL-8 levels predict early metastatic spread of breast cancer. The purpose of this study was to test our hypothesis that tumors that overexpress COX-2 induce IL-8 production., Materials and Methods: We cotransfected MCF-10A (nonmalignant breast epithelial) cells, as well as MDA-231 (highly metastatic human breast cancer) cell lines with a pSG5-COX-2 vector and pEF1a-Luc-IRES-Neo vector (luciferase reporter). COX-2 overexpression was confirmed by Western blot and PGE2 (a product of the COX-2 pathway) immunoassay. IL-8 production was measured by immunoassay. In vivo testing used a nude mouse model to measure COX-2 and IL-8 production from breast cancer cells that had metastasized to bone (bone-seeking clones (BSCs)). Long bone metastases were localized and quantified by luciferase imaging (Xenogen IVIS system) and X-ray. BSCs were isolated and cultured and then tested for the production of PGE2 and IL-8., Results: COX-2 overexpression caused a 4- to 5-fold increase in IL-8 production in both MCF-10A and MDA-231 cells in vitro. In vivo, we observed that the MDA-231-BSC (metastatic cells isolated from bone metastases) produced significantly greater levels of both PGE2 and IL-8 compared to the parental MDA-231 cells (P < 0.01). In contrast to the results obtained with these estrogen receptor-negative cell lines, COX-2 expression failed to induce IL-8 in the MCF-7 estrogen receptor-positive breast cancer cell line. Treatment with the COX-2 inhibitor NS-398 at a low 1-mu[scap]M dose reduced the production of IL-8 in COX-2-transfected MDA-231 cells by 30%, thus confirming the involvement of COX-2 in IL-8 induction., Conclusion: COX-2 expression induced formation of PGE2 and IL-8 in breast cancer cells. Since PGE2 and IL-8 stimulate osteoclasts to resorb bone, COX-2 inhibition is a potential target for treatment to prevent bone metastases.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to induce apoptosis, otherwise known as programmed cell death, in many malignant cells without any known detrimental effects to normal cells. These aspects of TRAIL indicate the potential of TRAIL as a therapeutic agent in cancer. Ovarian cancer remains the deadliest gynecologic malignancy and is the fourth leading cause of death due to cancer in women. However, it has been shown in studies that ovarian cancer cells are sensitive to TRAIL-induced cell death when treated with TRAIL alone or in combination with chemotherapeutic agents. TRAIL signals through two death receptors, TRAIL-R1 and TRAIL-R2, to induce apoptosis. TRAIL also binds to two other cell surface receptors, TRAIL-R3 and TRAIL-R4, which do not have intracellular death domains and therefore do not transmit the apoptotic signal upon ligation with TRAIL. It has been shown that a chemokine, interleukin-8 (IL-8), may play a role in ovarian tumor progression due to its elevated presence in the fluid surrounding ovarian cancer tissues. Possible roles for IL-8 in ovarian tumorigenesis include angiogenesis and metastasis. Because the mechanism of regulation for TRAIL-induced apoptosis needs to be clarified, the role of IL-8 in TRAIL-induced apoptosis of ovarian cancer cells was studied. Results showed that the presence of IL-8 regulates cell-surface expression of TRAIL receptors in ovarian cancer cell lines in vitro. There may be a role for the p38 mitogen-activated protein kinase (MAPK) pathway in TRAIL-induced apoptosis of ovarian cancer cell.

Extracellular matrix (ECM) remodeling is essential for normal development and tissue repair. Although many roles for extracellular proteinases in the breakdown of ECM have been established, the regulations of these proteinases in human tissue are not fully understood. Inflammatory cytokines have been implicated in the regulation of several matrix metalloproteinases. To determine whether these mediators have a similar effect on fibrinolysis and the remodeling of the fibrin provisional matrix, we examined the role of cytokines on the regulation of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) in human skin. In this report, we show that interleukin-8 (IL-8), but not other cytokines tested, is a potent inducer of the 38-kDa uPA in organ-cultured human skin. In addition, the uPA inhibitor, PAI-1, was not affected by IL-8. When primary epidermal human keratinocytes were treated with IL-8, 55-kDa pro-uPA was significantly induced in the conditioned medium. The mRNA expression of uPA in the keratinocytes was found to be constitutively elevated and was not affected by IL-8. To support such a notion, activation of the 5'-flanking promoter of the human uPA gene was measured using the CAT reporter assay. Consistent with the results of mRNA measurement, the promoter is constitutively active in keratinocytes and is not affected by IL-8. In contrast, the promoter construct is neither active in the dermal fibroblasts nor stimulated by the cytokine. This differential transactivation of uPA gene in these cells indicates that keratinocyte-specific factors may govern the basal expression of the gene. These results indicate a complex regulation of uPA expression in epidermal cells.

The aims of this study were to investigate the effects of Interleukin-8 (IL-8) and Growth related oncogene-alpha (Gro-alpha) on neutrophil apoptosis and determine the potential for a selective CXCR2 antagonist to inhibit these responses. IL-8 and Gro-alpha both produced dose dependent inhibition of spontaneous human neutrophil apoptosis after 16 hours incubation (59+/-3.5% and 27.5+/-3% respectively; EC50s 2.2+/-1.8 nM, and 0.5+/-0.2 nM respectively). The pro-survival effect of a fixed concentration of agonist (IL-8 or Gro-alpha) on cultured neutrophils was abrogated by a selective CXCR2 antagonist SB272844 (K(D)s 253 nM and 49.9 nM in the presence of IL-8 or Gro-alpha respectively). Our data suggests that the anti-apoptotic effect of Gro-alpha is mediated through CXCR2 as selective CXCR2 blockade with SB272844 can potently abrogate this response. The inhibitory effect of IL-8 may in addition partly be mediated through CXCR1 as SB272844 was less potent in its ability to abrogate the anti-apoptotic effects of IL-8 when this agent was used as an agonist. CXCR2 antagonists may have a therapeutic role in controlling neutrophil-driven inflammation by reducing neutrophil recruitment and restoring neutrophils to the tissue clearance pathway of apoptosis., (Copyright 2002 Elsevier Science Ltd.)

Background: Wound healing is a sequential biological process that involves the integration of chemotaxis of neutrophils, mitosis and migration of keratinocytes, and remodeling of the scar, all of which are regulated by specific soluble mediators. To modulate wound healing specific mediators have to be identified and functionally characterized. Therefore we addressed this study on the polymorphonuclear leukocyte (PMN) attractant interleukin-8 (IL-8) and its function in epidermal wound healing., Materials and Methods: Peptide purification, bioassays for PMN chemotaxis, and sequential IL-8 measurements were performed on human wound fluid from burn blisters and skin graft donor sites. Histology for IL-8 immunoreactivity was included. In vitro human keratinocytes were assayed for proliferation, migration, and integrin expression after IL-8 treatment. Wounding experiments with topical IL-8 were performed in a chimeric mouse model., Results: IL-8 was found to be the major bioactive chemoattractant for PMNs in human blister and skin graft donor site wound fluids (mean levels ranging from 173 ng/ml Postoperative Day (POD) 1 to 2130 ng/ml (POD 5)). Released intracellular epidermal IL-8 immunoreactivity at the wound edge was considered as an immediate source of IL-8 while NH(2)-terminal analysis revealed the 77-amino-acid residue form as a second source of IL-8 possibly PMN derived. In vitro experiments on the effect of recombinant human (rh) IL-8 on keratinocyte proliferation revealed a rise in cell number (4.8-fold, ED(50) = 0.6 ng/ml), which was accompanied by an increase in cells in S phase and overexpression of the integrin subunit alpha6. In vivo topically applied IL-8 (1 microg/ml) on human skin grafts in a chimeric mouse model enhanced reepithelialization in IL-8 treated animals over controls due to elevated numbers of mitotic keratinocytes. Wound contraction was significantly diminished by topical IL-8., Conclusions: These results indicate the sequential function of endogenous IL-8 in all phases of human wound healing. Topical IL-8 may be useful in impaired wound healing., (Copyright 2000 Academic Press.)

Background: Dysregulated neutrophil (PMN) apoptosis is thought to contribute to an exaggerated inflammatory response in diseases such as acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS). The CXC chemokines, interleukin-8 (IL-8) and growth-related oncogene alpha (Gro-alpha), contribute to the inflammatory response and suppress PMN apoptosis. We hypothesized that PMN generation of CXC chemokines is an autocrine/paracrine mechanism for amplification of the PMN inflammatory response via suppression of apoptosis., Methods: Freshly isolated human PMNs from healthy donors were incubated with IL-8 or Gro-alpha (100 ng/ml) for 0-12 h, and apoptosis was analyzed at 24 h. De novo synthesis of IL-8 or Gro-alpha was measured using an ELISA. To determine if receptors were available to bind these newly synthesized ligands (125)I radiolabeled monoclonal antibodies specific for each receptor (CXCRI, CXCRII) were used to determine PMN receptor density. Comparison was by one-way ANOVA., Results: Significant suppression of apoptosis was seen at 24 h with only 4 h exposure to IL-8 or Gro-alpha (n = 5, P < 0.05). PMNs cultured with IL-8 for 4 h produced 31 +/- 4.3 ng/ml IL-8 by 24 h; PMNs cultured with Gro-alpha produced 19.7 +/- 4.0 ng/ml Gro-alpha (n = 6, P < 0. 05). Neither chemokine induced significant production of the other chemokine. The addition of either ligand promoted upregulation of CXCR1 (n = 4, P < 0.05) at 24 h. However, CXCR2 was downregulated by Gro-alpha and IL-8 to 71 +/- 7.5 and 79 +/- 6.3% of control, respectively (P < 0.05)., Conclusion: IL-8 and Gro-alpha, which suppress apoptosis, stimulate their own production after short-term incubation with PMNs. PMNs maintain the ability to respond to these chemokines through expression of the CXC receptors which suggests that PMNs are active participants in the suppression of apoptosis at inflammatory sites. CXCRI remains upregulated after prolonged stimulation and may be an important target for mediating neutrophil responses to IL-8., (Copyright 2000 Academic Press.)

It is reported repeatedly that severe hepatocellular necrosis along with infiltration of neutrophils occurs after acute cadmium exposure. Neutrophils, which migrate by the gradient of chemoattractants such as interleukin-8, are believed to play an important role in inflammation at the damaged sites. To investigate whether neutrophils aggravate or repair the liver injury induced by cadmium, we checked the hepatotoxic effects of cadmium on human interleukin-8 transgenic mice (hIL-8Tg), which overexpressed IL-8 and displayed an inability of neutrophil migration resulting from both the lack of chemotactic gradient and the downregulation of l-selectin on the surface of neutrophils. A significantly lower survival rate was observed in hIL-8Tg compared with wild-type mice after subcutaneous administration of cadmium. Evident liver injury characterized by abrupt increases in plasma GOT and GPT levels was found in hIL-8Tg at 18 h after cadmium administration. Histological examinations, including H & E staining and esterase staining, revealed the infiltration of numerous neutrophils into the damaged liver tissues in wild-type mice, and the inhibition of the neutrophil migration into the liver as well as enhanced hepatocellular necrosis in hIL-8Tg. Peripheral white blood cell and polymorphonuclear cell counts increased and reached their peaks at 12 h after cadmium administration in wild-type mice, whereas the increase in blood leukocyte counts was delayed in hIL-8Tg. There was no significant difference in the amounts of cadmium accumulated in liver and kidneys between wild-type mice and hIL-8Tg. In conclusion, an acute cadmium hepatotoxic effect was exacerbated in hIL-8Tg resulting from inhibited neutrophil migration, suggesting that migrated neutrophils can prevent aggravation of liver injury by acute cadmium administration., (Copyright 2000 Academic Press.)

In this study we demonstrate that mice which lack the murine interleukin-8 receptor homologue exhibit enhanced resistance during the early stage of infection (1-4 days after i.v. challenge with Listeria monocytogenes). This result is surprising in that interleukin-8 and other CXC chemokines are key players in the accumulation of inflammatory neutrophils, which is thought to be critical for resistance to listeriosis. Paradoxically, some of the interleukin-8 receptor knockout mice that survived acute infection with L. monocytogenes demonstrated evidence of chronic infection with L. monocytogenes., (Copyright 1998 Academic Press.)

We investigated the effect of the extracellular matrix proteins fibronectin (Fn) and laminin (Ln) on polymorphonuclear leukocytes (PMN) bactericidal activity. Adherence of PMN to increasing concentrations of Ln significantly increased the killing of Escherichia coli after 240 min of adherence, while fibronectin significantly increased PMN staphlacidal activity after 240 min of adherence. The addition of IL-1beta and IL-8 but not TNF-alpha increased PMN bactericidal activity against E. coli when PMN were adhered to Ln, while TNF-alpha and IL-8 increased PMN bactericidal activity against Staphylococcus aureus when PMN were adhered to Ln. TNF-alpha increased PMN killing of E. coli when PMN were adhered to Fn, while only IL-1beta increased the killing of S. aureus when PMN were adhered to FN. Anti-VLA-3 (alpha3/beta1) monoclonal antibodies (mAbs) inhibited the effect of Ln on PMN bactericidal activity, while anti-VLA-5 (alpha5/beta1) mAbs inhibited the effect of Fn on PMN bactericidal activity. Progressive cross-linkage of these two receptors led to a dose-dependent reduction in PMN bactericidal activity for both pathogens when PMN were adhered to Ln or Fn, respectively. These results demonstrate that extracellular matrix proteins +/- exogenously added cytokines have the capacity to regulate PMN bactericidal activity. The signals sent by these matrix proteins to increase PMN bactericidal activity are transduced primarily via separate alpha subunits of the beta1 integrin complex. Stimulation of these receptors might lead to potential upregulation of PMN bactericidal activity which would be potentially advantageous in vivo at sites of infection., (Copyright 1997 Academic Press.)

In studies of disease processes, increasing knowledge leads to an increased awareness of the complexity of the underlying mechanisms. The intense research activity in the chemokine field has made this acutely manifest. Numerous chemokines have been discovered through the use of (1) bioassay of in vitro cell culture supernatants and in vivo exudates from animal models of inflammation and (2) molecular biology techniques. Any one chemokine can often be produced by a number of different cell types and exert its effects on different target cells. This has been interpreted by some as implying a high degree of redundancy. Although this is understandable, in disease processes parallel and sequential mechanisms are possible, and potentially important therapeutic targets have emerged. There is compelling evidence from animal and clinical studies that eosinophils are important effector cells in asthma, but this relationship is as yet unproven in the human disease. Two possible targets to prevent eosinophil recruitment to the lung are IL-5 and its receptor, which are important in several aspects of eosinophil biology, and eotaxin and its receptor, CCR3. The eotaxin receptor is particularly attractive as a target as it is expressed in high numbers on eosinophils, but not other leukocytes, and appears to be the major detector of the eosinophil for eotaxin and other chemokines such as MCP-4. Eotaxin and CCR3 knockout mice are being developed, and animal models will continue to be invaluable when antagonists are available. In the shape of receptor antagonists, the chemokine field may yet provide the final proof of concept for the long-established eosinophil theory of asthma in humans.

T-cell infiltration into synovium is a crucial process for rheumatoid arthritis (RA). To investigate the mechanism of T-cell infiltration, we studied T-cell attracting activity in synovial tissue extracts of RA or osteoarthritis (OA) whose synovium lacks T-cell accumulation. RA extracts attracted twofold more T cells than OA extracts. By gel filtration column chromatography the activity of RA extracts was separated into two peaks; one was eluted at the 67-kDa region and the other was at the 12-kDa region, while the latter was absent in OA extracts. The activity eluted at the 12-kDa region was absorbed mostly by an antibody against IL-8/NAP-1, a potent T-cell chemotactic factor. IL-8/NAP-1 concentrations in RA extracts were much higher than those in OA extracts and correlated to T-cell attracting activity eluted at the 12-kDa region. The checkerboard analysis revealed that the 67-kDa activity was chemokinetic but not chemotactic. These results suggest that IL-8/NAP-1 is the major T-cell chemoattractant in RA-synovium.

To determine whether neural crest-derived neuroblastoma cells may release cytokines which regulate the function of leukocytes, we found that neuroblastoma (HTB-11) cells did not constitutionally express IL-1 beta, TNF alpha, or IL-8 mRNA. However, TNF alpha, which induced HTB-11 cells to differentiate to perineurium-like cells, induced expression of IL-8 mRNA in a dose- and time-dependent fashion. In contrast, pentoxifylline (1 mM), which promoted HTB-11 cells to differentiate to polygonal neuron-like cells, did not induce IL-8 mRNA expression. As determined by enzyme-linked immunoassay, high levels of IL-8 were detectable in the culture supernatants from TNF alpha-treated neuroblastoma cells, but not pentoxifylline-treated neuroblastoma cells (19.60 +/- 2.34 vs 0.10 +/- 0.06 ng/ml). Culture supernatants obtained from TNF alpha-treated neuroblastoma cells induced chemotaxis of neutrophils and lymphocytes that was significantly blocked by anti-IL-8 neutralizing antibodies. Detection of a leukocyte chemotactic factor was not observed in the culture supernatants from pentoxifylline-treated cells. These results suggest that neural crest-derived perineurium-like cells, but not neuron-like cells, may release a leukocyte chemotactic factor or factors such as IL-8 which could be involved in leukocyte recruitment seen in inflammatory diseases affecting peripheral nerves.