302 Journal of Leukocyte Biology Volume 59, February 1996 Endogenously produced urokinase amplifies tumor necrosis factor-a secretion by THP-1 mononuclear phagocytes Robert G. Sitrin, Susan B. Shoilenberger, Robert M. Strieter, and Margaret R. Gyetko Puiriwnary and Critical Care Medicine Divtsion, Department ofinternal Medicine, University of Michigan, Ann Arbor Abstract: This study examined the effects of endo- genous urokinase (uPA) on lipopolysaccharide (LPS)-stimulated tumor necrosis factor a (TNF-a) secretion in THP-1 mononuclear phagocytes. Anti- uPA monoclonal antibody (mAb) suppressed LPS- driven TNF-czsecretion by 61.6 ± 5.9% (P < .001), and PAl-i, a uPA inhibitor, suppressed it to 53.1 ± 8.2% ofthe control value (P < .001). Up-regulation of TNF-cx mRNA was suppressed in parallel with secreted TNF-cz protein. TNF-a secretion was unaf- fected by depleting plasminogen or by aprotinin, a plasmin inhibitor. When endogenous uPA was dis- placed from the cell, exogenous high-molecular- weight (intact) uPA augmented LPS-driven TNF-cc 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-a neosynthesis of LPS-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. J. Leukoc. Rio!. 59: 302-311; 1996. Key Words: macrophage . plasminogen activator cytokine lipopolysaccharide INTRODUCTION Proteases are integral to inflammatory responses, not only by contributing to tissue injury and remodeling but also by regulating the activation and function of inflammatory cells. Mononuclear phagocytes (Ms) use the plasminogen activator system as an important mechanism for regulating the environment in the inflammatory milieu [1, 2]. These cells synthesize and secrete urokinase-type plasminogen activator (uPA) and PA inhibitors (principally PAI-2) and express specific high-affinity uPA receptors on the plasma membrane (uPAR; CD87) [1, 2]. Cytokines stimulate neo- synthesis of all elements of the PA-plasmin system, and in some cases there is significant cytokine specificity [1, 3, 4]. The role of the uPA-plasmin system in M function has been only partially elucidated, but it appears to be particu- larly important for tissue invasion, chemotaxis, and remod- eling extracellular matrix proteins [2, 5-8]. Beyond the effect of these enzymes on stromal integrity, evidence is accumulating that uPA and plasmin are involved in regu- lating the release of cytokines or activation of cytokines from latent forms [9-11]. In addition, uPA can regulate leukocyte differentiation and activation by mechanisms quite distinct from those of its catalytic functions. In neu- trophils, binding uPA to plasma membrane uPAR triggers signal transduction to the cell interior, as evidenced by calcium fluxes and protein tyrosine phosphorylation [12, 13]. Engagement of uPAR by uPA enhances uPAR-medi- ated adhesion to vitronectin and differentiation of M-like cells [14, 15]. In some instances, the amino terminus of uPA (bearing the uPAR binding domain) is sufficient to trigger a response, and uPA enzymatic activity, located in the carboxyl terminus, is entirely unnecessary [13, 15]. In neutrophils, both the amino and carboxyl terminal regions of uPA must interact with partner proteins to initiate signal transduction [12]. Thus, distinct elements of the uPA structure can subserve diverse mechanisms for modulating leukocyte function. One of the difficulties inherent in studying the uPA- plasmin system in cell culture is that M4s produce suffi- cient PA inhibitors to complicate experimental manipulation of uPA function [2]. One means of circum- venting this problem is to study the THP-1 human mono- cytic leukemia line. THP-1 cells, which have been used extensively as models of Ms, express uPA and uPAR normally but produce an inactive form of PAI-2 [16, 17]. As a result, functional PA activity is readily measured in cell supernatants. In this study, we sought to determine whether the PA-plasmin system affects tumor necrosis fac- tor cx (TNF-a) secretion by Ms, using THP-1 cells as a Abbreviations: ATF, amino terminal fragment; FBS, fetal bovine se- rum; FITC, fluorescein isothiocyanate; HMW, high molecular weight; IgG, immunoglobulin G; IL-8, interleukin-8; LMW, low molecular weight; LPS, lipopolysaccharide; mAb, monoclonal antibody; M, mononuclear phagocyte; mPU, milliPloug unit; PLG, plasminogen; SDS, sodium dodecyl sulfate; TNF-a, tumor necrosis factor a; uPA, urokinase- type plasminogen activator; uPAR, uPA receptor; UV, ultraviolet; Z- LTBE, thiobenzyl benzyloxycarbonyl-L-lysinate. Reprint requests: Robert G. Sitrin. Pulmonary and Critical Care Medi- cine Division, 3916 Taubman Center, Box 0360, Ann Arbor, MI 48109- 0360. Received April 7, 1995; revised October 4, 1995; accepted October 6, 1995.