Endogenously produced urokinase amplifies tumor necrosis factor‐α secretion by THP‐1 mononuclear phagocytes

This study examined the effects of endogenous urokinase (uPA) on lipopolysaccharide (LPS)‐stimulated tumor necrosis factor α (TNF‐α) secretion in THP‐1 mononuclear phagocytes. Anti‐uPA monoclonal antibody (mAb) suppressed LPS‐driven TNF‐α secretion by 61.6 ± 5.9% (P < .001), and PAI‐1, a uPA inhibitor, suppressed it to 53.1 ± 8.2% of the control value (P < .001). Up‐regulation of TNF‐α mRNA was suppressed in parallel with secreted TNF‐α protein. TNF‐α secretion was unaffected by depleting plasminogen or by aprotinin, a plasmin inhibitor. When endogenous uPA was displaced from the cell, exogenous high‐molecular‐weight (intact) uPA augmented LPS‐driven TNF‐α secretion. By contrast, a uPA fragment containing the catalytic domain was inhibitory, and the uPA receptor‐binding domain had no effect. We conclude that endogenous uPA amplifies TNF‐α neosynthesis of UPS‐stimulated THP‐1 mononuclear phagocytes. The effect requires intact uPA and is independent of plasmin activity. This represents a novel mechanism by which a mononuclear phagocyte–derived protease contributes to generating proinflammatory signals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142208/1/jlb0302.pd

Reduced 5-lipoxygenase metabolism of arachidonic acid in macrophages rrom 1,25-dihydroxyvitamin D3-deficient rats

The peripheral blood monocyte (PBM) migrates into tissues and differentiates into mature tissue macrophages. Previous investigations from our laboratory have demonstrated that PBM have reduced 5-lipoxygenase (5-LO) metabolism of arachidonic acid (AA) and 5-LO activating protein (FLAP) expression as compared to differentiated alveolar macrophages (AM). Moreover, PBM differentiated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) displayed increased leukotriene synthesis and a parallel increase in FLAP expression. In the present study, we sought to examine the physiological role of 1,25-(OH)2D3 in the regulation of eicosanoid metabolism in terminally differentiated alveolar and peritoneal macrophages (PM), utilizing a well characterized rat model of vitamin D3-deficiency. AM from vitamin D3-deficient rats demonstrated reduced 5-LO metabolism of AA and a parallel reduction in FLAP expression compared to control rats. Similarly, PM from vitamin D3-deficient rats demonstrated reduced 5-LO metabolism of AA. The effect of vitamin D3 was specific for the 5-LO pathway, not affecting total release of AA or its metabolism via 12-lipoxygenase or cyclooxoygenase (COX) pathways in macrophages. Furthermore, it did not affect COX protein expression in macrophages or type II alveolar epithelial cells. In control animals, 1,25-(OH)2D3 concentrations were greater in bronchoalveolar lavage fluid (2.,6-fold) and peritoneal lavage fluid (1.6-fold) than in serum, which may account for the greater FLAP expression in AM and PM than in PBM. These observations suggest that 1,25-(OH)2D3 plays a physiological role in upregulating the 5-LO pathway in tissue macrophages in vivo.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31212/1/0000114.pd

The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1β mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system

Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung

A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain. Expression of genes known to regulate vascular permeability was modulated by LeTx in the lungs of the more susceptible A/J strain. Unexpectedly, the largest set of genes with altered expression was immune specific, characterized by the up-regulation of lymphoid genes and the down-regulation of myeloid genes. Transcripts encoding neutrophil chemoattractants, modulators of tumor regulation and angiogenesis were also differentially expressed in both mouse strains. These studies provide new directions for the investigation of vascular leakage and pulmonary edema induced by anthrax LeTx

Differential Expression of Cytokines in Response to Respiratory Syncytial Virus Infection of Calves with High or Low Circulating 25-Hydroxyvitamin D3

Deficiency of serum levels of 25-hydroxyvitamin D3 has been related to increased risk of lower respiratory tract infections in children. Respiratory syncytial virus (RSV) is a leading cause of low respiratory tract infections in infants and young children. The neonatal calf model of RSV infection shares many features in common with RSV infection in infants and children. In the present study, we hypothesized that calves with low circulating levels of 25-hydroxyvitamin D3 (25(OH)D3) would be more susceptible to RSV infection than calves with high circulating levels of 25(OH)D3. Calves were fed milk replacer diets with different levels of vitamin D for a 10 wk period to establish two treatment groups, one with high (177 ng/ml) and one with low (32.5 ng/ml) circulating 25(OH)D3. Animals were experimentally infected via aerosol challenge with RSV. Data on circulating 25(OH)D3 levels showed that high and low concentrations of 25(OH)D3 were maintained during infection. At necropsy, lung lesions due to RSV were similar in the two vitamin D treatment groups. We show for the first time that RSV infection activates the vitamin D intracrine pathway in the inflamed lung. Importantly, however, we observed that cytokines frequently inhibited by this pathway in vitro are, in fact, either significantly upregulated (IL-12p40) or unaffected (IFN-γ) in the lungs of RSV-infected calves with high circulating levels of 25(OH)D3. Our data indicate that while vitamin D does have an immunomodulatory role during RSV infection, there was no significant impact on pathogenesis during the early phases of RSV infection. Further examination of the potential effects of vitamin D status on RSV disease resolution will require longer-term studies with immunologically sufficient and deficient vitamin D levels