Urokinase expression in mononuclear phagocytes: cytokine‐specific modulation by interferon‐γ and tumor necrosis factor‐α

This study delineates the regulatory effects of inflammatory cytokines on mononuclear phagocyte plasminogen activator (PA) activity. The mechanisms by which mononuclear phagocytes modulate PA activity are described. Mononuclear phagocytes regulate net PA activity by the balanced expression of urokinase‐type PA (uPA), in either secreted or membrane‐associated forms, and a specific plasminogen activator inhibitor, PAI‐2. Therefore, understanding how immunomodulators regulate macrophage PA activity requires that the comparative effects of uPA and PAI‐2 be elucidated. We determined how recombinant interferon‐γ (IFN) and tumor necrosis factor‐α (TNF) regulate plasminogen activation in monoblast‐like U937 cells and normal human monocytes. In U937 cells, both IFN and TNF induced concurrent increases in secreted PA and PA inhibitor activities. These effects were accompanied by increased immunoreactive uPA and PAI‐2 in conditioned media (enzyme‐linked immunosorbent assay) and steady‐state levels of cellular uPA and PAI‐2 mRNA (Northern analysis). To determine the relative abilities of IFN and TNF to either promote or inhibit plasmin generation, we directly compared the effects IFN and TNF, using optimal stimulating concentrations. IFN induced PA activity to 180% of the level achieved by TNF. In contrast, IFN elicited only 78% of the PA inhibitor produced by TNF stimulation. These differences in secreted activity can be explained by the shift in balance between uPA and PAI‐2 proteins. Immunoreactive uPA was induced equally by IFN and TNF, but TNF generated higher levels of PAI‐2. The same overall pattern of results was seen in normal human monocytes. IFN and TNF differ greatly in the ability to augment receptor‐bound PA activity in U937 cells, as IFN induced a twofold increase but TNF had no effect. We conclude that IFN and TNF modulate mononuclear phagocyte proteolytic activity through coordinate regulation of secreted and receptor‐bound uPA, balanced against concurrent expression of PAI‐2. These effects are cytokine specific, as IFN is superior to TNF in stimulating expression of both secreted and receptor‐associated PA activities. These properties suggest mechanisms by which mononuclear phagocytes control proteolysis in cytokinerich inflammatory foci.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141202/1/jlb0256.pd

Monocyte urokinase expression: modulation by interleukins

This study delineates the regulatory effect of interleukin‐1 (IL‐1) and interleukin‐2 (IL‐2) on monocyte plasminogen activator (PA) activity. Mononuclear phagocytes regulate net PA activity by modulating the expression of urokinase‐type PA (uPA) and a specific plasminogen activator inhibitor, PA1‐2. To understand the regulation of mononuclear phagocyte PA activity, it is important to compare the expression of uPA and PAI‐2. In this study, we determined the relative abundance of secreted PA and PA inhibitor activity in human monocyte‐conditioned medium after stimulation with human recombinant IL‐1 or IL‐2. In agreement with our previous description of tumor necrosis factor‐α and interferon‐ γ stimulation of mononuclear phagocytes, we found no detectable PA activity in conditioned medium. Both IL‐1 and IL‐2 had dose‐dependent effects, significantly up‐ regulating PA inhibitor activity in monocyte‐conditioned medium (up to 11‐fold). To further investigate the mechanism underlying this effect, Northern blot analysis was done to measure steady‐state mRNA for uPA and PAI‐2. Consistent with the increase in secreted PA inhibitor activity, we found that both IL‐1 and IL‐2 significantly increased steady‐state mRNA for PAI‐2. In addition, however, both IL‐1 and IL‐2 increased steady‐state mRNA for uPA. IL‐1 appears to increase mRNA for uPA to a greater extent than does IL‐2. We conclude that IL‐1 and IL‐2 modulate monocyte proteolytic activity by increasing expression of uPA and PAI‐2 with a resultant predominance of PAI‐2. We further conclude that cytokine‐specific regulation of plasminogen activity is achieved partly by varying the proportionate expression of uPA and PAI‐2.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141561/1/jlb0598.pd

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

Monocyte 1α‐hydroxylase regulation: induction by inflammatory cytokines and suppression by dexamethasone and uremia toxin

Abstract: Alveolar macrophages acquire lα‐hydroxylase activity in inflammation, and thereby metabolize 25 hydroxyvitamin D3 (25 D3) to the active metabolite, la,25‐dihydroxyvitamin D3 (1,25 D3, calcitriol). Cal‐ citriol is a potent differentiation agent that modulates mononuclear phagocyte activation and effector functions. The mediators that induce macrophage lα‐hydroxylase activity are not well delineated. Furthermore, it is unclear whether calcitriol is a product only of terminally differentiated macrophages or whether less mature mononuclear phagocytes can produce it as well. The ability of newly recruited monocytes to produce calcitriol as an autocrine differentiation agent is particularly important in inflammation, as it may substantially expand the functional repertoire of these cells. To assess the effects of cytokines on lα‐hydroxylase activity, blood monocytes were cultured in the presence and absence of human recombinant tumor necrosis factor a (TNF‐α), interferon‐7 (IFN‐γ), and interleukins 1 and 2 and then incubated with 25 D3 substrate. The conditioned media were assayed for calcitriol by high‐performance liquid chromatography and competitive receptor binding assay. No detectable calcitriol was produced by unstimulated monocytes. However, all the cytokines markedly increased monocyte calcitriol production (range 133‐151 pg/mg protein; in all cases P < .001). We then determined whether calcitriol production was suppressed by preincubation with either dexamethasone or the putative uremia toxin guanidinosuccinic acid (GSA). Dexamethasone pretreatment significantly inhibited subsequent cytokine‐induced calcitriol production by monocytes, as did GSA (average 69 and 63% of control, respectively).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141704/1/jlb0017.pd

Calcitriol-mediated modulation of urokinase-type plasminogen activator and plasminogen activator inhibitor-2

Calcitriol-induced differentiation of U937 mononuclear phagocytes is known to have divergent effects on the synthesis of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). In this study, we sought to determine whether calcitriol affects the expression of these proteins by modulating intermediate signal trasduction involving intracellular calcium and protein kinase C (PKC). U937 cells were stimulated with calcitriol (50 nM) for 6-72 hr, inducing a transient increase in specific binding of [3H]phorbol dibutyrate ([3H]PDBu), seen only after 24 hr. Staurosporine (2 nM), a PKC inhibitor, had no effect on calcitriol-induced secretion of plasminogen activator (PA) activity. However, staurosporine significantly (P &lt; 0.05) inhibited the ability of calcitriol to enhance phorbol myristate acetate (PMA)-induced secretion of PA inhibitor activity, indicating that this priming effect of calcitriol requires expression of PKC. The calcium ionophore A23187 (0.1 [mu]M) induced a modest increase in secreted PA inhibitor activity, in contrast to the secretion of PA activity which is consistently seen in response to calcitriol. Northern blot analysis demonstrated that A23187 induced an increase in PAI-2 mRNA and a marked reduction in uPA mRNA, while calcitriol induced opposite changes in both mRNA species. We conclude that calcitriol modulates uPA and PAI-2 expression by multiple mechanisms that are both PKC dependent and PKC independent. Our studies also demonstrated that increased intracellular calcium alters the synthesis of both uPA and PAI-2 in a manner which favors expression of PA inhibitor activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29456/1/0000538.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

Activation of Urokinase Plasminogen Activator and Its Receptor Axis Is Essential for Macrophage Infiltration in a Prostate Cancer Mouse Model1

Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA) and its receptor (uPAR) axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role inmonocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT), uPA-/-, and uPAR-/- mice. Tumor volume was significantly diminished in both uPA-/- and uPAR-/- mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA-/- mice compared with uPAR-/- mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA-/- and uPAR-/- mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA-/- and uPAR-/- mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3+ cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors