The comparative release of FGF1 by hypoxia and temperature stress.

The signal peptide-less FGF gene family prototype, FGF1 is released in response to temperature stress in vitro as a latent reducing agent-sensitive homodimer non-covalently complexed with the extravesicular p40 domain of p65 synaptotagmin (Syt)1. Because FGF1 is well recognized as an angiogenesis factor in vivo and angiogenesis is known to be induced by hypoxia, we examined the release of FGF1 and p40 Syt1 under conditions of hypoxia and temperature stress using a chemostatic microcarrier cell culture system. We report that like the pathway used by FGF1 and p40 Syt1 release under temperature stress, hypoxia also induces the release of FGF1 and p40 Syt1 with similar kinetic and pharmacologic properties including the requirement for functional cysteine residues. Lastly, FGF1 and p40 Syt1 release in response to hypoxia and temperature stress is sensitive to lipoxygenase and cyclooxygenase inhibitors suggesting that arachidonic acid metabolism may play an important role in the mechanism of FGF1 release in vitro.

The precursor but not the mature form of IL1alpha blocks the release of FGF1 in response to heat shock.

Interleukin (IL)1alpha mediates proinflammatory events through its extracellular interaction with the IL1 type I receptor. However, IL1alpha does not contain a conventional signal peptide sequence that provides access to the endoplasmic reticulum-Golgi apparatus for secretion. Thus, we have studied the release of the precursor (p) and mature (m) forms of IL1alpha from NIH 3T3 cells. We have demonstrated that mIL1alpha but not pIL1alpha was released in response to heat shock with biochemical and pharmacological properties similar to those reported for the stress-mediated release pathway utilized by fibroblast growth factor (FGF)1. However, unlike the FGF1 release pathway, the IL1alpha release pathway appears to function independently of synaptotagmin (Syt)1 because the expression of a dominant-negative form of Syt1, which represses the release of FGF1, did not inhibit the release of mIL1alpha in response to temperature stress. Interestingly, whereas the expression of both mIL1alpha and FGF1 in NIH 3T3 cells did not impair the stress-induced release of either polypeptide, the expression of both pIL1alpha and FGF1 repressed the release of FGF1 in response to temperature stress. These data suggest that the release of mIL1alpha requires proteolytic processing of its precursor form and that mIL1alpha and FGF1 may utilize similar but distinct mechanisms for export.

Experimental reovirus serotype 1/strain Lang infection of the lung: a model for the study of the lung in the context of mucosal immunity.

A number of studies have examined the nature of the respiratory immune response to particular pathogens. Although many pathogens stimulate specific immunity in the lung, they frequently are not effective immunogens at other mucosal sites. Because the gastrointestinal tract is a major inductive site for mucosal immunity, a pathogen that is an effective respiratory and gut immunogen would allow studies of the interaction of the lung with gut mucosal immune system. Reovirus, a respiratory isolate that previously has been shown to be an effective gut mucosal immunogen, provides a potential model of the relationship of the lung to the gut mucosal immune system. In this report, we demonstrate that intranasal application of reovirus serotype 1/strain Lang (1/L) to CD-1 mice elicits an acute lymphocytic inflammatory infiltration of the lung and hyperplasia of the lung-associated lymph nodes. The initial inflammatory response occurs in the airspaces and interstitium of the lung. As the infection progresses, the initially diffuse cellular infiltrate becomes more focused around small bronchioles. Viral replication occurs predominantly during the first week of the infection, and infectious virions are eliminated during the second week. After the elimination of infectious virions, a secondary response consisting of the appearance of plasma cells adjacent to pulmonary arteries develops as the primary infiltrate organizes into peribronchiolar follicles, resembling the human inflammatory lung condition termed follicular bronchiolitis. These two infiltration patterns were also observed by immunohistochemical analysis of the the infected lung. Whereas CD4+ and CD8+ lymphocytes and Mac-1+ cells were found to be more closely associated with the primary infiltration process, B220+ lymphocytes were observed adjacent to pulmonary arteries. These results establish respiratory reovirus 1/L infection as a viable model for future investigations of the mucosal immune response in the lung and its relationship to the common mucosal immune system.

Prolonged induction of IL-8 gene expression in a human fibroblast cell line infected with reovirus serotype 1 strain Lang.

Viruses which infect mucosal surfaces commonly infect these particular anatomical sites based on both the virion structure and the interaction of the virus with a particular microenvironment. We infected a human lung epithelial cell line, a human gut epithelial cell line, and a human lung fibroblast cell line with reovirus 1/L to explore how this natural isolate of both the lung and the gut may interact with mucosal surfaces. While reovirus infection of the gut and lung epithelial cell lines was lytic, a chronic infection was established in the human lung fibroblast cell line. All three cell lines also produced interleukin-8 (IL-8) after infection with reovirus 1/L, and IL-8 production was not dependent upon viral replication. A prolonged production of IL-8 was observed in the chronically infected lung fibroblast cell line, suggesting that this mucosal population may be involved in the generation of inflammatory responses after the resolution of the initial lytic infection of the epithelium. These studies provide an in vitro model system for analyzing the interaction of reovirus 1/L with resident mucosal cell populations.

Respiratory-mucosal lymphocyte populations induced by reovirus serotype 1 infection.

Respiratory virus infections are a serious health challenge. While models exist to study immune mechanisms of the respiratory tract, they have not allowed analysis of the interaction of the lower respiratory tract with other components of the mucosal immune system. This study demonstrates that reovirus 1/Lang, an effective gut mucosal immunogen, also provides a useful model of respiratory mucosal infection. Intra-nasal infection of Balb/c mice resulted in severe viral bronchopneumonia. Major components of the cellular inflammatory response in the lung interstitium and alveolar spaces were CD8 lymphocytes. Lung lymphocyte populations exhibited lysis of reovirus-infected, but not uninfected target cells after in vitro culture. The GCT antigen, a germinal center B-cell and CD8 T-cell marker, was present on 21-60% of the inflammatory lymphocytes. A novel population of GCT-expressing CD4+ lymphocytes unique to reovirus-stimulated lung alveolar and interstitial lymphocyte populations was identified.

Copper induces the assembly of a multiprotein aggregate implicated in the release of fibroblast growth factor 1 in response to stress.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.

S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro.

S100A13, a member of the S100 gene family of Ca(2+)-binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37 degrees C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer.

Respiratory reovirus 1/L induction of intraluminal fibrosis. A model for the study of bronchiolitis obliterans organizing pneumonia.

Bronchiolitis obliterans organizing pneumonia (BOOP) is a term that was first applied in 1985 to describe a long-observed but unclassified pattern of acute lung injury. BOOP lesions are characterized by fibrous extensions into the alveolar spaces in association with a peribronchiolar organizing pneumonia. Since 1985, an increasing number of reports of BOOP have appeared in the clinical literature, and it is now accepted that BOOP is a significant pulmonary syndrome. Although BOOP can be associated with a number of documented pulmonary insults, many cases are not associated with known causes and are thus classified as idiopathic. The lack of an appropriate small animal model that closely mimics the generation of BOOP lesions has been an impediment to basic studies of the pathogenic mechanisms responsible for the generation of BOOP in humans. In this report, we describe an animal model for BOOP in which CBA/J mice infected with reovirus serotype 1/strain Lang develop BOOP lesions. These lesions closely resemble those seen in humans and occur in a well defined temporal sequence that proceeds from initial peribronchiolar inflammatory lesions to characteristic, fibrotic cellular BOOP lesions over a 3-week time course.