Fimbria-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells

Epidemiological studies support that chronic periodontal infections are associated with an increased risk of cardiovascular disease. Previously, we reported that the periodontal pathogen Porphyromonas gingivalis accelerated atherosclerotic plaque formation in hyperlipidemic apoE-/- mice, while an isogenic fimbria-deficient (FimA-) mutant did not. In this study, we utilized 41 kDa (major) and 67 kDa (minor) fimbria mutants to demonstrate that major fimbria are required for efficient P. gingivalis invasion of human aortic endothelial cells (HAEC). Enzyme-linked immunosorbent assay (ELISA) revealed that only invasive P. gingivalis strains induced HAEC production of pro-inflammatory molecules interleukin (IL)-1beta, IL-8, monocyte chemoattractant protein (MCP)-1, intracellular adhesion molecule (ICAM)-1, vascular cellular adhesion molecule (VCAM)-1 and E-selectin. The purified native forms of major and minor fimbria induced chemokine and adhesion molecule expression similar to invasive P. gingivalis, but failed to elicit IL-1beta production. In addition, the major and minor fimbria-mediated production of MCP-1 and IL-8 was inhibited in a dose-dependent manner by P. gingivalis lipopolysaccharide (LPS). Both P. gingivalis LPS and heat-killed organisms failed to stimulate HAEC. Treatment of endothelial cells with cytochalasin D abolished the observed pro-inflammatory MCP-1 and IL-8 response to invasive P. gingivalis and both purified fimbria, but did not affect P. gingivalis induction of IL-1beta. These results suggest that major and minor fimbria elicit chemokine production in HAEC through actin cytoskeletal rearrangements; however, induction of IL-1beta appears to occur via a separate mechanism. Collectively, these data support that invasive P. gingivalis and fimbria stimulate endothelial cell activation, a necessary initial event in the development of atherogenesis.     

Induction of monocyte chemoattractant protein-1 by Porphyromonas gingivalis in human endothelial cells

The association between periodontal and cardiovascular diseases could be mediated by direct interaction of periodontal pathogens with cardiac tissue. In order to explore this possibility, the effect of the periodontal pathogen Porphyromonas gingivalis on monocyte chemoattractant protein-1 (MCP-1) production by endothelial cells was investigated. When incubated with live P. gingivalis 381, MCP-1 production by human umbilical vein endothelial cells (HUVEC) was potently increased. Compared to the type strain 381, non-adhesive/invasive strains (W50 and DPG3) did not increase MCP-1 production, which was also demonstrated at the mRNA level. Killed P. gingivalis 381 was much less effective than live bacteria for MCP-1 induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, prevented MCP-1 mRNA up-regulation by P. gingivalis 381, suggesting that internalization of P. gingivalis is necessary for MCP-1 induction. In conclusion, the secretion of high levels of MCP-1 resulting from interactions of P. gingivalis with endothelial cells could enhance atherosclerosis progression by contributing to the recruitment of monocytes.     

Modulation of cytokine production by Porphyromonas gingivalis in a macrophage and epithelial cell co-culture model

Epithelial cells and macrophages play a major role in the host response to Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. Secretion of high levels of cytokines by these cells is believed to contribute to periodontal tissue destruction. To investigate the interactions between P. gingivalis and these two major cell types, we characterized the production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and regulated on activation normal T cell expressed and secreted (RANTES) by an in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 cell line) following a challenge with different strains of P. gingivalis. P. gingivalis cells stimulated the secretion of pro-inflammatory cytokines (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) in the co-culture model. Responses to P. gingivalis infection were influenced by the macrophage/epithelial cell ratios of the cultures. In addition, the level of secretion of these inflammatory mediators was dependent on the bacterial strain and the multiplicity of infection (MOI) used. The use of a gingipain-deficient mutant of P. gingivalis or the addition of a cysteine protease inhibitor suggested that the level of cytokines secreted by the co-culture model was underestimated due to an extensive proteolytic degradation. This study showed that P. gingivalis can modulate the levels of inflammatory mediators, which may contribute to the progression of periodontitis.     

Lysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.     

The role of the chemokines MCP-1, GRO-alpha, IL-8 and their receptors in the adhesion of monocytic cells to human atherosclerotic plaques

Monocyte adhesion to the arterial endothelium and subsequent migration into the intima are central events in the pathogenesis of atherosclerosis. Previous experimental models have shown that chemokines can enhance monocyte–endothelial adhesion by activating monocyte integrins. Our study assesses the role of chemokines IL-8, MCP-1 and GRO-α, together with their monocyte receptors CCR2 and CXCR2 in monocyte adhesion to human atherosclerotic plaques. In an adhesion assay, a suspension of monocytic U937 cells was incubated with human atherosclerotic artery sections and the levels of endothelial adhesion were quantified. Adhesion performed in the presence of a monoclonal antibody to a chemokine, chemokine receptor or of an isotype matched control immunoglobulin, shows that antibodies to all chemokines tested, as well as their receptors, inhibit adhesion compared to the control immunoglobulins. Immunohistochemistry demonstrated the expression of MCP-1, GRO-α and their receptors in the endothelial cells and intima of all atherosclerotic lesions. These results suggest that all these chemokines and their receptors can play a role in the adhesion of monocytes to human atherosclerotic plaques. Furthermore, they suggest that these chemokine interactions provide potential targets for the therapy of atherosclerosis.     

Statins affect the presentation of endothelial chemokines by targeting to multivesicular bodies

BACKGROUND: In addition to lowering cholesterol, statins are thought to beneficially modulate inflammation. Several chemokines including CXCL1/growth-related oncogene (GRO)-α, CXCL8/interleukin (IL)-8 and CCL2/monocyte chemoattractant protein (MCP)-1 are important in the pathogenesis of atherosclerosis and can be influenced by statin-treatment. Recently, we observed that atorvastatin-treatment alters the intracellular content and subcellular distribution of GRO-α in cultured human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the mechanisms involved in this phenomenon. METHODOLOGY/ PRINCIPAL FINDINGS: The effect of atorvastatin on secretion levels and subcellular distribution of GRO-α, IL-8 and MCP-1 in HUVECs activated by interleukin (IL)-1β were evaluated by ELISA, confocal microscopy and immunoelectron microscopy. Atorvastatin increased the intracellular contents of GRO-α, IL-8, and MCP-1 and induced colocalization with E-selectin in multivesicular bodies. This effect was prevented by adding the isoprenylation substrate GGPP, but not the cholesterol precursor squalene, indicating that atorvastatin exerts these effects by inhibiting isoprenylation rather than depleting the cells of cholesterol. CONCLUSIONS/ SIGNIFICANCE: Atorvastatin targets inflammatory chemokines to the endocytic pathway and multivesicular bodies and may contribute to explain the anti-inflammatory effect of statins at the level of endothelial cell function.     

Treponema denticola induces interleukin-8 and macrophage chemoattractant protein 1 production in human umbilical vein epithelial cells

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.     

TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1

The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-alpha in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-alpha induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-alpha activation. When IL-10, a suppressive cytokine, was incubated with TNF-alpha and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.     

The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.     

Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.     

Wide proinflammatory effect of electronegative low-density lipoprotein on human endothelial cells assayed by a protein array

Electronegative low-density lipoprotein (LDL(-)) is a modified subfraction of LDL present in plasma able to induce the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) by human umbilical vein endothelial cells (HUVEC). To ascertain whether further inflammation mediator release could be induced by LDL(-), a protein array system was used to measure 42 cytokines and related compounds. Native LDL and LDL(-) isolated from normolipemic subjects were incubated for 24 h with HUVEC and culture supernatants were used to measure inflammation mediator release. The protein array revealed that IL-6, granulocyte/monocyte colony-stimulating factor (GM-CSF) and growth-related oncogene (GRO) release were increased by cultured HUVEC in response to LDL(-). LDL(-) enhanced production of IL-6 (4-fold vs. LDL(+)), GM-CSF (4-fold), GRObeta (2-fold) and GROgamma (7-fold) was confirmed by ELISA. Time-course experiments revealed that IL-6 was released earlier than the other inflammation mediators, suggesting a first-wave cytokine action. However, the addition of IL-6 alone did not stimulate the production of IL-8, MCP-1 or GM-CSF. Moreover, IL-8, MCP-1 or GM-CSF alone did not promote the release of the other inflammatory molecules. Modification of LDL(+) by phospholipase A(2)-mediated lipolysis or by loading with non-esterified fatty acids (NEFA) reproduced the action of LDL(-), thereby suggesting the involvement of NEFA and/or lysophosphatidylcholine in the release of these molecules. Our results indicate that LDL(-) promotes a proinflammatory phenotype in endothelial cells through the production of cytokines, chemokines and growth factors.     

Induction and Secretion of the Chemokines Interleukin-8 and Monocyte Chemotactic Protein-1 in Human Immature Leukemia Cell Lines

We investigated expression and secretion of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in human myeloid cell lines. Quantitative determination by ELISA revealed a significant constitutive production of both chemokines in the cell lines HL-60 and NB-4 (>1000 pg/ml IL-8 and >400 pg/ml MCP-1 per million cells), while in the cell lines EOL-1, KASUMI-1 and KG-1 only 10–100 pg/ml IL-8 and MCP-1 were detected. Tetradecanoyl phorbol acetate (TPA) strongly increased the IL-8 and MCP-1 amounts in the culture supernatants of all five cell lines. The TPA-induced NB-4 produced the largest amounts of both chemokines (>40,000 pg/ml). The strongest induction was seen in EOL-1 (>100-fold increase). Besides TPA, tumor necrosis factor-α (TNFα) also distinctively enhanced IL-8 and MCP-1 production. The calcium ionophore A-23187 and thapsigargin, an inhibitor of the Ca²⁺-ATPase, differentially induced IL-8 and MCP-1 secretion in the cell lines investigated, suggesting that, at least in some cell lines, intracellular free Ca²⁺ might be important for chemokine secretion. Dexamethasone significantly prevented the IL-8 and MCP-1 production of stimulated cells, emphasizing the potent anti-inflammatory property of glucocorticoids. Similarly, the protein kinase inhibitor staurosporine clearly decreased the TPA-induced chemokine secretion in NB-4 cells, indicating the involvement of protein kinases in the signal transduction pathway which leads to enhanced chemokine secretion.     

Different chemokines are expressed in human arthritic bone biopsies: IFN-gamma and IL-6 differently modulate IL-8, MCP-1 and rantes production by arthritic osteoblasts

In the present study we analyse chemokine expression in the remodelling of subchondral bone in arthritis patients. Trabecular bone biopsies were tested by immunohistochemistry to identify interleukin (IL)-8, GRO-alpha, MCP-1, RANTES, MIP-1alpha and MIP-1beta expression. Subsequently, we evaluated by immunoassay the effect of interferon (IFN)-gamma and IL-6 on chemokine production by osteoarthritis (OA), rheumatoid arthritis (RA) and post-traumatic (PT) patients' isolated osteoblasts (OB). OB constitutively produced in situ IL-8, GRO-alpha, MCP-1, RANTES and MIP-1alpha. MIP-1beta was positive only in mononuclear cells. In RA many of these chemokines were also produced by mononuclear cells. IFN-gamma significantly down-regulated IL-8 and up-regulated MCP-1 produced by OB from all patients tested, whereas it did not affect the other chemokines analysed. Moreover, IFN-gamma reduced IL-1beta-stimulated IL-8 production but significantly increased both MCP-1 and RANTES. Interestingly, IL-6 significantly downregulated IFN-gamma-induced MCP-1 production, that was significantly lower in OA compared to RA patients. OB expressed chemokines both in vivo and in vitro suggesting that these cells are primary effectors in the bone capable of regulating autocrine/paracrine circuits that affect bone remodelling in these diseases.     

Effects of Antioxidant and Nitric Oxide on Chemokine Production in TNF-α-stimulated Human Dermal Microvascular Endothelial Cells

Chemokines have been implicated convincingly in the driving of leukocyte emigration in different inflammatory reactions. Multiple signaling mechanisms are reported to be involved in intracellular activation of chemokine expression in vascular endothelial cells by various stimuli. Nevertheless, redox-regulated mechanisms of chemokine expression in human dermal microvascular endothelial cells (HDMEC) remain unclear. This study examined the effects of pyrrolidine dithiocarbamate (PDTC, 0.1?mM) and spermine NONOate (Sper-NO, 1?mM) on the secretion and gene expression of chemokines, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, regulated upon activation normal T cell expressed and secreted (RANTES), and eotaxin. This study also addresses PDTC and Sper-NO effects on activation of nuclear factor kappa B (NF-κB) induced by TNF-α (10?ng/ml). Treatment with TNF-α for 8?h significantly increased secretion of IL-8, MCP-1, and RANTES, but not of eotaxin, in cultured HDMEC. Up-regulation of these chemokines was suppressed significantly by pretreatment with PDTC or Sper-NO for 1?h, but not by 1?mM 8-bromo-cyclic GMP. The mRNA accumulation of IL-8, MCP-1, RANTES, and eotaxin, and activation of NF-κB were induced by TNF-α for 2?h; all were suppressed significantly by the above two pretreatments. These findings indicate that both secretion and mRNA accumulation of IL-8, MCP-1, and RANTES in HDMEC induced by TNF-α are inhibited significantly by pretreatment with PDTC or Sper-NO, possibly via blocking redox-regulated NF-κB activation. These results suggest that restoration of the redox balance using antioxidant agents or nitric oxide pathway modulators may offer new opportunities for therapeutic interventions in inflammatory skin diseases.     

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Synovial fluid from patients with various arthritides contains procoagulant, cell-derived microparticles. Here we studied whether synovial microparticles modulate the release of chemokines and cytokines by fibroblast-like synoviocytes (FLS). Microparticles, isolated from the synovial fluid of rheumatoid arthritis (RA) and arthritis control (AC) patients (n = 8 and n = 3, respectively), were identified and quantified by flow cytometry. Simultaneously, arthroscopically guided synovial biopsies were taken from the same knee joint as the synovial fluid. FLS were isolated, cultured, and incubated for 24 hours in the absence or presence of autologous microparticles. Subsequently, cell-free culture supernatants were collected and concentrations of monocyte chemoattractant protein-1 (MCP-1), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1) were determined. Results were consistent with previous observations: synovial fluid from all RA as well as AC patients contained microparticles of monocytic and granulocytic origin. Incubation with autologous microparticles increased the levels of MCP-1, IL-8 and RANTES in 6 of 11 cultures of FLS, and IL-6, ICAM-1 and VEGF in 10 cultures. Total numbers of microparticles were correlated with the IL-8 (r = 0.91, P < 0.0001) and MCP-1 concentrations (r = 0.81, P < 0.0001), as did the numbers of granulocyte-derived microparticles (r = 0.89, P < 0.0001 and r = 0.93, P < 0.0001, respectively). In contrast, GM-CSF levels were decreased. These results demonstrate that microparticles might modulate the release of chemokines and cytokines by FLS and might therefore have a function in synovial inflammation and angiogenesis.     

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.