The viral CC chemokine-binding protein vCCI inhibits monocyte chemoattractant protein-1 activity by masking its CCR2B-binding site

Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site.     

Role of monocyte chemotactic protein-1/CC chemokine ligand 2 on gamma delta T lymphocyte trafficking during inflammation induced by lipopolysaccharide or Mycobacterium bovis bacille Calmette-Guérin

Gammadelta T lymphocytes are involved in a great variety of inflammatory and infectious responses. However, the mechanisms by which gammadelta T lymphocytes migrate to inflamed sites are poorly understood. In this study we investigate the role of monocyte chemotactic protein (MCP)-1 in regulating gammadelta T cell migration after LPS or Mycobacterium bovis bacille Calmette-Guérin (BCG) challenge. LPS-induced gammadelta T cell influx was significantly inhibited by either pretreatment with dexamethasone or vaccinia virus Lister 35-kDa chemokine binding protein, vCKBP, a CC chemokine neutralizing protein, suggesting a role for CC chemokines in this phenomenon. LPS stimulation increased the expression of MCP-1 mRNA and protein at the inflammation site within 6 h. It is noteworthy that LPS was unable to increase MCP-1 production or gammadelta T cell recruitment in C3H/HeJ, indicative of the involvement of Toll-like receptor 4. Gammadelta T cells express MCP-1 receptor CCR2. Pretreatment with anti-MCP-1 mAb drastically inhibited LPS-induced in vivo gammadelta T cell mobilization. Indeed, MCP-1 knockout mice were unable to recruit gammadelta T cells to the pleural cavity after LPS stimulation, effect that could be restored by coadministration of MCP-1. In addition, BCG-induced gammadelta lymphocyte accumulation was significantly reduced in MCP-1 knockout mice when compared with wild-type mice. In conclusion, our results indicate that LPS-induced gammadelta T lymphocyte migration is dependent on Toll-like receptor 4 and sensitive to both dexamethasone and CC chemokine-binding protein inhibition. Moreover, by using MCP-1 neutralizing Abs and genetically deficient mice we show that LPS- and BCG-induced gammadelta T lymphocyte influx to the pleural cavity of mice is mainly orchestrated by the CC chemokine MCP-1.     

Structure and function of A41, a vaccinia virus chemokine binding protein

The vaccinia virus (VACV) A41L gene encodes a secreted 30 kDa glycoprotein that is nonessential for virus replication but affects the host response to infection. The A41 protein shares sequence similarity with another VACV protein that binds CC chemokines (called vCKBP, or viral CC chemokine inhibitor, vCCI), and strains of VACV lacking the A41L gene induced stronger CD8+ T-cell responses than control viruses expressing A41. Using surface plasmon resonance, we screened 39 human and murine chemokines and identified CCL21, CCL25, CCL26 and CCL28 as A41 ligands, with Kds of between 8 nM and 118 nM. Nonetheless, A41 was ineffective at inhibiting chemotaxis induced by these chemokines, indicating it did not block the interaction of these chemokines with their receptors. However the interaction of A41 and chemokines was inhibited in a dose-dependent manner by heparin, suggesting that A41 and heparin bind to overlapping sites on these chemokines. To better understand the mechanism of action of A41 its crystal structure was solved to 1.9 A resolution. The protein has a globular beta sandwich structure similar to that of the poxvirus vCCI family of proteins, but there are notable structural differences, particularly in surface loops and electrostatic charge distribution. Structural modelling suggests that the binding paradigm as defined for the vCCI-chemokine interaction is likely to be conserved between A41 and its chemokine partners. Additionally, sequence analysis of chemokines binding to A41 identified a signature for A41 binding. The biological and structural data suggest that A41 functions by forming moderately strong (nM) interactions with certain chemokines, sufficient to interfere with chemokine-glycosaminoglycan interactions at the cell surface (microM-nM) and thereby to destroy the chemokine concentration gradient, but not strong enough to disrupt the (pM) chemokine-chemokine receptor interactions.     

Comprehensive mapping of poxvirus vCCI chemokine-binding protein. Expanded range of ligand interactions and unusual dissociation kinetics.

In compiling a comprehensive map of the ligand binding capacity of elements within the chemokine system, we have determined the spectrum of chemokines capable of interacting with the poxvirus-encoded viral CC chemokine inhibitor, vCCI. More than 80 chemokines were tested in parallel for their ability to displace radiolabeled signature chemokines from vCCI. Of these chemokines, 26 showed potential high affinity interactions. These interactions revealed an expanded spectrum of binding capacity for vCCI to now include molecules such as human myeloid progenitor inhibitory factor-1 as ligands. In addition, high affinity viral protein-protein interactions were revealed. For example, binding between poxvirus vCCI and the herpesvirus vMIP-II from HHV8 occurs with IC(50) approximately 10-50 nm. Unusual dissociation kinetics were observed between certain chemokines and vCCI. Notably, many ligands displayed a precipitous displacement profile, suggesting marked positive cooperativity of binding. Finally, heterologous competition provided evidence for overlapping but distinct binding sites for the many chemokines that bind to vCCI. The determination of the binding fingerprint and unusual binding interactions of vCCI with a large number of chemokines suggest a finely honed evolutionary strategy of chemokine sequestration during viral infection.     

Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

An ectromelia virus protein that interacts with chemokines through their glycosaminoglycan binding domain

Poxviruses encode a number of secreted virulence factors that modulate the host immune response. The vaccinia virus A41 protein is an immunomodulatory protein with amino acid sequence similarity to the 35-kDa chemokine binding protein, but the host immune molecules targeted by A41 have not been identified. We report here that the vaccinia virus A41 ortholog encoded by ectromelia virus, a poxvirus pathogen of mice, named E163 in the ectromelia virus Naval strain, is a secreted 31-kDa glycoprotein that selectively binds a limited number of CC and CXC chemokines with high affinity. A detailed characterization of the interaction of ectromelia virus E163 with mutant forms of the chemokines CXCL10 and CXCL12α indicated that E163 binds to the glycosaminoglycan binding site of the chemokines. This suggests that E163 inhibits the interaction of chemokines with glycosaminoglycans and provides a mechanism by which E163 prevents chemokine-induced leukocyte migration to the sites of infection. In addition to interacting with chemokines, E163 can interact with high affinity with glycosaminoglycan molecules, enabling E163 to attach to cell surfaces and to remain in the vicinity of the sites of viral infection. These findings identify E163 as a new chemokine binding protein in poxviruses and provide a molecular mechanism for the immunomodulatory activity previously reported for the vaccinia virus A41 ortholog. The results reported here also suggest that the cell surface and extracellular matrix are important targeting sites for secreted poxvirus immune modulators.     

Structural Insights into the Interaction between a Potent Anti-inflammatory Protein,Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine,Eotaxin-1

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.     

Fas ligation and tumor necrosis factor α activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.     

Chemokines accumulate in the lungs of rats with severe pulmonary embolism induced by polystyrene microspheres

Pulmonary thromboembolism (PEm) is a serious and life threatening disease and the most common cause of acute pulmonary vascular occlusion. Even following successful treatment of PEm, many patients experience long-term disability due to diminished heart and lung function. Considerable damage to the lungs presumably occurs due to reperfusion injury following anti-occlusive treatments for PEm and the resulting chronic inflammatory state in the lung vasculature. We have used a rat model of irreversible PEm to ask whether pulmonary vascular occlusion in the absence of reperfusion is itself sufficient to induce an inflammatory response in lungs. By adjusting the severity of the vascular occlusion, we were able to generate hypertensive and nonhypertensive PEm, and then examine lung tissue for expression of CXC and C-C chemokine genes and bronchoalveolar lavage (BAL) fluid for the presence of chemokine proteins. Hypertensive and nonhypertensive PEm resulted in increased expression of both CXC and C-C chemokines genes in lung tissues. Hypertensive PEm was also associated with a 50-100-fold increase in protein content in lung BAL fluid, which included the CXC chemokines cytokine-induced neutrophil chemoattractant and macrophage-inflammatory protein 2. The presence of chemokines in BALs was reflected by a potent neutrophil chemotactic activity in in vitro chemotaxis assays. Abs to cytokine-induced neutrophil chemoattractant blocked the in vitro neutrophil chemotactic activity of BAL by 44%. Our results indicate that the ischemia and hypertension associated with PEm are sufficient to induce expression of proinflammatory mediators such as chemokines, and establish a proinflammatory environment in the ischemic lung even before reperfusion.     

The purified 14-kilodalton envelope protein of vaccinia virus produced in Escherichia coli induces virus immunity in animals.

Vaccinia virus (VV) was successfully used as a live vaccine to eradicate smallpox, but the nature of viral proteins involved in eliciting viral immunity has not yet been identified. A potential candidate is a 14-kDa VV envelope protein that is involved in virus penetration at the level of virus-cell fusion, in cell-cell fusion late in infection, and in virus dissemination. The 14-kDa envelope protein has been produced in Escherichia coli, with properties similar to those of the native protein found in the virus particle and in infected cells (C. Lai, S. Gong, and M. Esteban, J. Biol. Chem. 256:22174-22180, 1990). In this investigation, we showed that mice immunized with purified VV 14-kDa protein synthesized in E. coli in the form of a monomer or a trimer develop high-titer neutralizing antibodies and are protected when challenged with lethal doses of wild-type VV. Our findings demonstrate that it is possible to confer protection against VV through immunization with the 14-kDa envelope protein.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Membrane-bound CC chemokine inhibitor 35K provides localized inhibition of CC chemokine activity in vitro and in vivo

CC chemokines mediate mononuclear cell recruitment and activation in chronic inflammation. We have shown previously that gene transfer using recombinant adenoviruses, encoding a soluble CC chemokine-binding protein of vaccinia virus 35K, can dramatically reduce atherosclerosis and vein graft remodeling in apolipoprotein E knockout mice. In this study, we report the development of a membrane-bound form of 35K (m35K), tagged with GFP, which allows for localized, broad-spectrum CC chemokine blockade. In vitro experiments indicate that m35K-expressing cells no longer undergo CC chemokine-induced chemotaxis, and m35K-expressing cells can locally deplete the CC chemokines RANTES (CCL5) and MIP-1alpha (CCL3) from supernatant medium. This sequestration of CC chemokines can prevent chemotaxis of bystander cells to CC, but not CX(3)C chemokines. Intraperitoneal injection of mice with an adenovirus-encoding m35K leads to a significant (44%) decrease in leukocyte recruitment into the peritoneal cavity in a sterile peritonitis model. Intravenous adenovirus-encoding m35K delivery leads to m35K expression in hepatocytes, which confers significant protection against liver damage (75% reduction in liver enzymes) in a Con A-induced hepatitis model. In summary, we have generated a membrane-bound CC chemokine-binding protein (m35K) that provides localized broad-spectrum CC chemokine inhibition in vitro and in vivo. m35K may be a useful tool to study the role of CC chemokines in leukocyte trafficking and block the recruitment of monocytes in chronic inflammation.     

Monocyte chemoattractant protein-1 mediates cockroach allergen-induced bronchial hyperreactivity in normal but not CCR2-/- mice: the role of mast cells.

Bronchial eosinophil and mononuclear cell infiltrates are a hallmark of the asthmatic lung and are associated with the induction of reversible airway hyperreactivity. In these studies, we have found that monocyte chemotactic protein-1 (MCP-1), a CC (beta) chemokine, mediates airway hyperreactivity in normal and allergic mice. Using a murine model of cockroach Ag-induced allergic airway inflammation, we have demonstrated that anti-MCP-1 Abs inhibit changes in airway resistance and attenuate histamine release into the bronchoalveolar lavage, suggesting a role for MCP-1 in mast cell degranulation. In normal mice, instillation of MCP-1 induced prolonged airway hyperreactivity and histamine release. In addition, MCP-1 directly induced pulmonary mast cell degranulation in vitro. These latter effects would appear to be selective because no changes were observed when macrophage-inflammatory protein-1alpha, eotaxin, or MCP-3 were instilled into the airways of normal mice or when mast cells were treated in vitro. Airway hyperreactivity was mediated by MCP-1 through CCR2 because allergen-induced as well as direct MCP-1 instilled-induced changes in airway hyperreactivity were significantly attenuated in CCR2 -/- mice. The neutralization of MCP-1 in allergic animals and instillation of MCP-1 in normal animals was related to leukotriene C4 levels in the bronchoalveolar lavage and was directly induced in pulmonary mast cells by MCP-1. Thus, these data identify MCP-1 and CCR2 as potentially important therapeutic targets for the treatment of hyperreactive airway disease.     

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.     

Influenza A virus infection inhibits the efficient recruitment of Th2 cells into the airways and the development of airway eosinophilia

Most infections with respiratory viruses induce Th1 responses characterized by the generation of Th1 and CD8(+) T cells secreting IFN-gamma, which in turn have been shown to inhibit the development of Th2 cells. Therefore, it could be expected that respiratory viral infections mediate protection against asthma. However, the opposite seems to be true, because viral infections are often associated with the exacerbation of asthma. For this reason, we investigated what effect an influenza A (flu) virus infection has on the development of asthma. We found that flu infection 1, 3, 6, or 9 wk before allergen airway challenge resulted in a strong suppression of allergen-induced airway eosinophilia. This effect was associated with strongly reduced numbers of Th2 cells in the airways and was not observed in IFN-gamma- or IL-12 p35-deficient mice. Mice infected with flu virus and immunized with OVA showed decreased IL-5 and increased IFN-gamma, eotaxin/CC chemokine ligand (CCL)11, RANTES/CCL5, and monocyte chemoattractant protein-1/CCL2 levels in the bronchoalveolar lavage fluid, and increased airway hyperreactivity compared with OVA-immunized mice. These results suggest that the flu virus infection reduced airway eosinophilia by inducing Th1 responses, which lead to the inefficient recruitment of Th2 cells into the airways. However, OVA-specific IgE and IgG1 serum levels, blood eosinophilia, and goblet cell metaplasia in the lung were not reduced by the flu infection. Flu virus infection also directly induced AHR and goblet cell metaplasia. Taken together, our results show that flu virus infections can induce, exacerbate, and suppress features of asthmatic disease in mice.     

Attenuation of a vaccine strain of vaccinia virus via inactivation of interferon viroceptor

BACKGROUND: Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-gamma receptor homolog (viroceptor). METHODS: To facilitate the monitoring of virus properties, first we constructed a Lister vaccine strain derivative VV-RG expressing optical reporters. Further, we constructed a VV-RG derivative, VV-RG8, which lacks the IFN-gammaR viroceptor (B8R gene product). Replication, immunological and pathogenic properties of the constructed strains were compared. RESULTS: Viruses did not show significant differences in humoral and cellular immune responses of immune-competent mice. Replication of constructed viruses was efficient both in vitro and in vivo, but showed marked difference in kinetics of propagation. In cultured CV-1 epithelial cells, the VV-RG8 strain retained the propagation potential of the parental virus, while, in the C6 glial cells, significant delay was observed in the kinetics of the VV-RG8 replication cycle compared to VV-RG. The pathogenesis of the viruses was tested by survival assay and biodistribution in nude mice. High dose inoculation of nude mice with VV-RG8 caused less pronounced virus dissemination, improved weight gain, and increased survival rate, as compared with the VV-RG strain. CONCLUSIONS: The replication-competent virus VV-RG8 carrying a mutation at the B8R gene is less pathogenic for mice than the parental vaccine virus. We anticipate that step-wise inactivation of VV vaccine genes involved in evasion of host immune response may provide an alternative approach for generation of hyper-attenuated replication-competent vaccines.     

Heterozygous defect in HIV-1 coreceptor CCR5 and chemokine production

CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus 1 (HIV-1). An inactive CCR5 allele with a 32-nucleotide deletion (CCR5Delta32) has been described that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. We found the allele CCR5Delta32 to be not rare in 399 Swiss blood donors with a frequency of 0.080. To assess the influence of defective CCR5 on production of its ligands we determined the capacity to produce the chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES in comparison with the production of the CXC chemokine IL-8 which does not bind to CCR5. Production of chemokines was determined during endotoxin stimulation of whole-blood samples ex vivo. Both, basal and LPS-induced chemokine production in 32 blood donors heterozygous for CCR5Delta32 were not significantly different when compared with 55 blood donors who were homozygous for the wild type CCR5 allele.     

Expression of secreted cytokine and chemokine inhibitors by ectromelia virus

The production of secreted proteins that bind cytokines and block their activity has been well characterized as an immune evasion strategy of the orthopoxviruses vaccinia virus (VV) and cowpox virus (CPV). However, very limited information is available on the expression of similar cytokine inhibitors by ectromelia virus (EV), a virulent natural mouse pathogen that causes mousepox. We have characterized the expression and binding properties of three major secreted immunomodulatory activities in 12 EV strains and isolates. Eleven of the 12 EVs expressed a soluble, secreted 35-kDa viral chemokine binding protein with properties similar to those of homologous proteins from VV and CPV. All of the EVs expressed soluble, secreted receptors that bound to mouse, human, and rat tumor necrosis factor alpha. We also detected the expression of a soluble, secreted interleukin-1beta (IL-1beta) receptor (vIL-1betaR) by all of the EVs. EV differed from VV and CPV in that binding of human (125)I-IL-1beta to the EV vIL-1betaR could not be detected. Nevertheless, the EV vIL-1betaR prevented the interaction of human and mouse IL-1beta with cellular receptors. There are significant differences in amino acid sequence between the EV vIL-1betaR and its VV and CPV homologs which may account for the results of the binding studies. The conservation of these activities in EV suggests evolutionary pressure to maintain them in a natural poxvirus infection. Mousepox represents a useful model for the study of poxvirus pathogenesis and immune evasion. These findings will facilitate future study of the role of EV immunomodulatory factors in the pathogenesis of mousepox.     

The vaccinia virus soluble alpha/beta interferon (IFN) receptor binds to the cell surface and protects cells from the antiviral effects of IFN

Poxviruses encode a broad range of proteins that interfere with host immune functions, such as soluble versions of receptors for the cytokines tumor necrosis factor, interleukin-1 beta, gamma interferon (IFN-gamma), IFN-alpha/beta, and chemokines. These virus-encoded cytokine receptors have a profound effect on virus pathogenesis and enable the study of the role of cytokines in virus infections. The vaccinia virus (VV) Western Reserve gene B18R encodes a secreted protein with 3 immunoglobulin domains that functions as a soluble receptor for IFN-alpha/beta. We have found that after secretion B18R binds to both uninfected and infected cells. The B18R protein present at the cell surface maintains the properties of the soluble receptor, binding IFN-alpha/beta with high affinity and with broad species specificity, and protects cells from the antiviral state induced by IFN-alpha/beta. VV strain Wyeth expressed a truncated B18R protein lacking the C-terminal immunoglobulin domain. This protein binds IFN with lower affinity and retains its ability to bind to cells, indicating that the C-terminal region of B18R contributes to IFN binding. The replication of a VV B18R deletion mutant in tissue culture was restricted in the presence of IFN-alpha, whereas the wild-type virus replicated normally. Binding of soluble recombinant B18R to cells protected the cultures from IFN and allowed VV replication. This represents a novel strategy of virus immune evasion in which secreted IFN-alpha/beta receptors not only bind the soluble cytokine but also bind to uninfected cells and protect them from the antiviral effects of IFN-alpha/beta, maintaining the cells' susceptibility to virus infections. The adaptation of this soluble receptor to block IFN-alpha/beta activity locally will help VV to replicate in the host and spread in tissues. This emphasizes the importance of local effects of IFN-alpha/beta against virus infections.     

Contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.