Inhalation of the crystalline form of silica is associated with a variety of pathologies from acute lung inflammation to silicosis, in addition to autoimmune disorders and cancer. Basic science researchers looking at the mechanisms involved with the earliest initiators of disease are focused on how the alveolar macrophage (AM) interacts with the inhaled silica particle and the consequences of silicainduced toxicity on the cellular level. Based on experimental results, several rationales have been developed on exactly how crystalline silica particles are toxic to the macrophage cell that is functionally responsible for clearance of the foreign particle. For example, silica is capable of producing reactive oxygen species (ROS) either directly (on the particle surface) or indirectly (produced by the cell as a response to silica) triggering cell-signaling pathways initiating cytokine release and apoptosis. With murine macrophages, reactive nitrogen species (RNS) are produced in the initial respiratory burst in addition to ROS. An alternative explanation for silica toxicity includes lysosomal permeability, where silica disrupts the normal internalization process leading to cytokine release and cell death. Still other research has focused on the cell surface receptors (collectively known as scavenger receptors) involved in silica binding and internalization. The silica-induced cytokine release and apoptosis is described as the function of receptor-mediated signaling rather than free radical damage. Current research ideas on silica toxicity and binding in the alveolar macrophage are reviewed and discussed.

Scavenger receptors (SR), on the surface of the macrophage, appear to be responsible for silica uptake and cell death signaling in the macrophage. The purpose of this study was to isolate which SRs (macrophage receptor with collagenous structure (MARCO), CD204, or CD36) were involved using a variety of SR single and double null mice. The findings indicated that MARCO was the critical SR involved in silica uptake and cytotoxicity in the primary alveolar macrophages (AM) from C57BL/6 mice, as there was no particle uptake or cell death in the absence of this SR. The level of MARCO expression on AM changed significantly with the absence of other SR, and silica uptake was proportional to cell surface MARCO expression. In addition, silica uptake and cytotoxicity were completely blocked by an anti-mouse MARCO antibody. Transfection of Chinese hamster ovary cells with human MARCO supported these conclusions, as silica particles bound to and initiated apoptosis in the MARCO-transfected cells. Strain differences with regard to SR distribution were also examined. There was a differential expression of these SR on AM from each strain, with MARCO dominant for C57BL/6, CD36 dominant on BALB/c, and all three SR expressed on 129/SvJ mice. Similar to the results with C57BL/6 AM, MARCO was involved with silica-induced cell death in the 129/SvJ strain. In contrast, BALB/c AM used an unidentified mechanism for silica uptake because the SR antibodies failed to block particle internalization. Taken together, these results indicate MARCO is the primary AM receptor interacting with silica, depending on mouse strain and level of constitutive expression.Inhaled silica particles are a known initiator of several human pathologies including silicosis, autoimmune disorders, and possibly even lung cancer (1, 2). The earliest contact with inhaled silica occurs when the alveolar macrophage (AM) 2 engulfs foreign material. This can lead to cell death in a fraction of the exposed cells, which may be the initial step in the disease process. Scavenger receptors (SR) on the surface of AM are one possible mechanism for the uptake and cell death signaling in the AM (3).SR are transmembrane glycoproteins found on macrophages, endothelial cells, and smooth muscle cells that bind to a number of ligands including Gram-negative bacteria, apoptotic cells, oxidized low-density lipoproteins, and polyinosinic acid to name just a few (4). The SR are divided into several different classes based on structure and function, but they all bind a broad range of ligands with polyanionic surface characteristics (5). The exact function of SR is still being determined, but they are believed to be an important feature of the innate immune response (6) and they are generally up-regulated in the presence of infection (7). Due to the promiscuous nature of these receptors, multiple functions have been suggested including, but not limited to, endocytosis followed by receptor recycling, cell adhesion (8), transcytosis, intracellular signaling (5), and uptake of particles with po...

Chronic exposure to crystalline silica can lead to the development of silicosis, an irreversible, inflammatory and fibrotic pulmonary disease. Although, previous studies established the macrophage receptor with collagenous structure (MARCO) as an important receptor for binding and uptake of crystalline silica particles in vitro, the role of MARCO in regulating the inflammatory response following silica exposure in vivo remains unknown. Therefore, we determined the role of MARCO in crystalline silica-induced pulmonary pathology using C57Bl/6 wild-type (WT) and MARCO(-/-) mice. Increased numbers of MARCO(+) pulmonary macrophages were observed following crystalline silica, but not phosphate-buffered saline and titanium dioxide (TiO(2)), instillation in WT mice, highlighting a specific role of MARCO in silica-induced pathology. We hypothesized that MARCO(-/-) mice will exhibit diminished clearance of silica leading to enhanced pulmonary inflammation and exacerbation of silicosis. Alveolar macrophages isolated from crystalline silica-exposed mice showed diminished particle uptake in vivo as compared with WT mice, indicating abnormalities in clearance mechanisms. Furthermore, MARCO(-/-) mice exposed to crystalline silica showed enhanced acute inflammation and lung injury marked by increases in early response cytokines and inflammatory cells compared with WT mice. Similarly, histological examination of MARCO(-/-) lungs at 3 months post-crystalline silica exposure showed increased chronic inflammation compared with WT; however, only a small difference was observed with respect to development of fibrosis as measured by hydroxyproline content. Altogether, these results demonstrate that MARCO is important for clearance of crystalline silica in vivo and that the absence of MARCO results in exacerbations in innate pulmonary immune responses.

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Alveolar macrophages (AM) in the lung have been documented to play pivotal roles in inflammation and fibrosis (silicosis) following inhalation of crystalline silica (CSiO(2)). In contrast, exposure to either titanium dioxide (TiO(2)) or amorphous silica (ASiO(2)) is considered relatively benign. The scavenger receptor macrophage receptor with collagenous structure (MARCO), expressed on AM, binds and internalizes environmental particles such as silica and TiO(2). Only CSiO(2) is toxic to AM, while ASiO(2) and TiO(2) are not. We hypothesize that differences in induction of pathology between toxic CSiO(2) and nontoxic particles ASiO(2) and TiO(2) may be related to their differential binding to MARCO. In vitro studies with Chinese hamster ovary (CHO) cells transfected with human MARCO and mutants were conducted to better characterize MARCO-particulate (ASiO(2), CSiO(2), and TiO(2)) interactions. Results with MARCO-transfected CHO cells and MARCO-specific antibody demonstrated that the scavenger receptor cysteine-rich (SRCR) domain of MARCO was required for particle binding for all the tested particles. Only TiO(2) required divalent cations (viz., Ca(+2) and/or Mg(+2)) for binding to MARCO, and results from competitive binding studies supported the notion that TiO(2) and both the silica particles bound to different motifs in SRCR domain of MARCO. The results also suggest that particle shape and/or crystal structure may be the determinants linking particle binding to MARCO and cytotoxicity. Taken together, these results demonstrate that the SRCR domain of MARCO is required for particle binding and that involvement of different regions of SRCR domain may distinguish downstream events following particle binding.

During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p<0.01). Flow cytometry data revealed a corresponding 4-fold increase in influx of neutrophils into the lungs by ss-Ino-RNA treatment. In our in vitro experiments, treatment of epithelial cells with ss-Ino-RNA reduced replication of respiratory syncytial virus (RSV). Interestingly, RNA structural analysis showed that ss-Ino-RNA had increased formation of secondary structures. Our data further revealed that extracellular ss-Ino-RNA was taken up by scavenger receptor class-A (SR-A) which activated downstream MAP Kinase pathways through Toll-like receptor 3 (TLR3) and dsRNA-activated protein kinase (PKR). Our data suggests that ss-Ino-RNA is an as yet undescribed virus-associated innate immune stimulus.

Propargyl alcohol (PA) is a high production volume chemical used in synthesis of many industrial chemicals and agricultural products. Despite the potential for prolonged or accidental exposure to PA in industrial settings, the toxicity potential of PA was not well characterized. To address the knowledge gaps relevant to the toxicity profile of PA, the National Toxicology Program (NTP) conducted 2-week, 14-week and 2-year studies in male and female F344/N rats and B6C3F1/N mice. For the 2-week inhalation study, the rats and mice were exposed to 0, 31.3, 62.5, 125, 250 or 500 ppm. Significant mortality was observed in both rats and mice exposed to ≥ 125 ppm of PA. The major target organ of toxicity in both mice and rats was the liver with exposure-related histopathological changes (250 and 500 ppm). Based on the decreased survival in the 2-week study, the rats and mice were exposed to 0, 4, 8, 16, 32 or 64 ppm of PA in the 14-week study. No treatment-related mortality was observed. Mean body weights of male (≥ 8 ppm) and female mice (32 and 64 ppm) were significantly decreased (7–16%). Histopathological changes were noted in the nasal cavity, and included suppurative inflammation, squamous metaplasia, hyaline droplet accumulation, olfactory epithelium atrophy, and necrosis. In the 2-year inhalation studies, the rats were exposed to 0, 16, 32 and 64 ppm of PA and the mice were exposed to 0, 8, 16 and 32 ppm of PA. Survival of male rats was significantly reduced (32 ppm and 64 ppm). Mean body weights of 64 ppm male rats were significantly decreased relative to the controls. Both mice and rats showed a spectrum of non-neoplastic changes in the nose. Increased neoplastic incidences of nasal respiratory/transitional epithelial adenoma were observed in both rats and mice. The incidence of mononuclear cell leukemia was significantly increased in male rats. In conclusion, the key findings from this study indicated that the nose was the primary target organ of toxicity for PA. Long term inhalation exposure to PA led to nonneoplastic changes in the nose, and increased incidences of respiratory/transitional epithelial adenomas in both mice and rats. Increased incidences of harderian gland adenoma may also have been related to exposure to PA in male mice.

Effective immunoglobulin responses play a vital role in protection against most pathogens. However, the molecular mediators and mechanisms responsible for signaling and selective expression of immunoglobulin types remain to be elucidated. Previous studies in our laboratory have demonstrated that protein kinase R (PKR) plays a crucial role in IgE responses to double-stranded RNA (dsRNA) in vitro. In this study, we show that PKR plays a critical role in IgG expression both in vivo and in vitro. PKR ؊/؊ mice show significantly altered serum IgG levels during respiratory syncytial virus (RSV) infection. IgG2a expression is particularly sensitive to a lack of PKR and is below the detection level in mock-or RSV-infected PKR ؊/؊ mice. Interestingly, we show that upon activation by anti-CD40 and gamma interferon (IFN-␥), B cells from PKR ؊/؊ mice show diminished major histocompatibility complex class II (MHC II), CD80, and CD86 levels on the cell surface compared to wild-type (WT) mice. Our data also show that PKR is necessary for optimal expression of adhesion molecules, such as CD11a and ICAM-1, that are necessary for homotypic aggregation of B cells. Furthermore, in this report we demonstrate for the first time that upon CD40 ligation, PKR is rapidly phosphorylated and activated, indicating that PKR is an early and novel downstream mediator of CD40 signaling pathways.