Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast.

Idiopathic Pulmonary Fibrosis (IPF) is characterized by injury and loss of lung epithelial cells, accumulation of fibroblasts/myofibroblasts and abnormal remodeling of the lung parenchyma. The prognosis for IPF patients is poor and current therapies are largely ineffective in preventing respiratory failure. Current therapeutic approaches target epithelial cell replacement, manipulation of fibroblasts/myofibroblasts, modulation of procoagulant/fibrinolytic activities, cytokine and growth factor production, angiogenesis, and reduction of oxidative stress. Myofibroblasts are the primary effector cells in fibrosis. These cells may be derived by the activation and proliferation of resident lung fibroblasts, from epithelial-mesenchymal transition (EMT), or through recruitment of circulating fibrocytes. Transforming growth factor beta (TGFbeta) is a profibrotic factor that increases fibroblast proliferation, stimulates the synthesis and deposition of connective tissue, and inhibits connective tissue breakdown. TGFbeta acts through the promoter of the type 1 collagen gene causing increased collagen synthesis. In addition, TGFbeta induces EMT in alveolar epithelial cells (AECs) in vitro and in vivo. AECs exhibit substantial plasticity and may serve as a source of fibroblasts and/or myofibroblasts in lung fibrosis. Therapeutic interventions interfering with the pathways that lead to myofibroblast expansion and AEC apoptosis should be of considerable benefit in the treatment of IPF. This review will focus on the critical role of TGFbeta on AECs EMT and myofibroblasts in the development of fibrosis.

Antigen-driven lymphocyte recruitment to the lung is diminished in the absence of urokinase-type plasminogen activator (uPA) receptor, but is independent of uPA.

The requirement for urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in T lymphocyte migration is unknown. uPA(-/-) mice have fewer pulmonary lymphocytes in response to certain infections, but its unknown whether this is due to diminished recruitment. Primed, recipient mice were IT inoculated with Ag. Three days later, fluorescently labeled lymphoblasts from background-matched control wild-type (WT), uPA(-/-), or uPAR(-/-) donor mice were injected i.v., and their recruitment was determined. Approximately twice the number of uPA(-/-) compared with WT lymphoblasts were recruited to the lungs of WT recipients. This difference was eliminated when uPA(-/-) and WT lymphoblasts were injected into uPA(-/-) recipients. Thus, the reduced number of lung lymphocytes in infected uPA(-/-) mice is not due to reduced recruitment. However, uPAR is critically involved in recruitment. Markedly fewer uPAR(-/-) compared with WT lymphoblasts were recruited to the lung. These findings suggest that uPAR may be a novel target for immune modulation in T lymphocyte-mediated disorders.

Urokinase receptor-deficient mice have impaired neutrophil recruitment in response to pulmonary Pseudomonas aeruginosa infection.

Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of beta2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a beta2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR-/-) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA-/- mice is indistinguishable from recruitment in WT mice. uPAR-/- mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR-/- neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.

Urokinase is required for T lymphocyte proliferation and activation in vitro.

We have previously demonstrated that urokinase-deficient (uPA-/-) mice do not increase lung T lymphocyte number and fail to mount protective immune responses during pulmonary Cryptococcus neoformans infection. These observations suggest a previously unconsidered role for urokinase-type plasminogen activator (uPA) in T lymphocyte-mediated immune responses. Accordingly, we sought to determine whether uPA is required for T cell receptor-mediated (TCR-mediated) lymphocyte proliferation and activation. Splenocytes from uPA-/- and uPA+/+ mice were stimulated with concanavalin A (Con A). The uPA-/- mice had diminished T cell proliferation as compared with uPA+/+ mice. Coculturing uPA-/- T cells with uPA+/+ accessory cells led to the restoration of proliferation. Similarly, T cell proliferation induced by CD3 cross-linking was diminished in uPA-/- mice as compared with uPA+/+ mice. T lymphocyte activation, defined as the induced expression of antigens and the elaboration of cytokines, was determined. The expression of CD69 and that of CD49d were diminished in response to Con A stimulation in uPA-/- mice as compared with uPA+/+ mice. The elaboration of cytokines in response to Con A was also altered in the uPA-/- mice. The production of the Th1 cytokines interferon-gamma and interleukin-12 was diminished in uPA-/- mice as compared with uPA+/+ mice. The uPA-/- mice produced increased amounts of interleukin-10, a Th2 cytokine. We conclude that the lack of uPA results in impaired T cell activation and proliferation in response to TCR-mediated signaling and the expression of a less Th1-polarized profile of cytokines. These findings suggest that the inability of uPA-/- mice to combat Cryptococcus neoformans infection may be caused by the impairment of T lymphocyte immune responses in the absence of uPA.

Urokinase-type plasminogen activator in inflammatory cell recruitment and host defense against Pneumocystis carinii in mice.

Effective host defense against Pneumocystis carinii depends upon the integrated actions of inflammatory cells and mediators in the lungs. Using immunocompetent and immunosuppressed mice, our laboratory has defined inflammatory changes in the lungs in response to P. carinii. However, the essential molecules and mechanisms required for cellular recruitment and for host defense against P. carinii are undefined. We hypothesized that urokinase-type plasminogen activator (uPA), a protease intimately involved in inflammatory cell migration and activation, is required for clearance of P. carinii. To test this hypothesis in vivo, we compared the intensity of P. carinii infection and inflammation in the lungs of mice lacking the uPA gene (uPA knockout mice) and in the lungs of wild-type mice. After intratracheal inoculation with P. carinii organisms, uPA knockout mice developed uniformly heavy P. carinii pneumonia while wild-type mice cleared the P. carinii inoculum. Bronchoalveolar lavage fluid from uPA knockout mice contained significantly smaller numbers of cells than did lavage fluid from wild-type mice. We conclude that deletion of the uPA gene prevents the clearance of P. carinii and reduces inflammatory cell recruitment. Therefore, uPA is an important participant in the network of inflammatory events required for the clearance of P. carinii, confirming an important role for this molecule in pulmonary host defense against opportunistic pathogens.

Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins.

During angiogenesis, endothelial cells penetrate fibrin barriers via undefined proteolytic mechanisms. We demonstrate that the fibrinolytic plasminogen activator (PA)-plasminogen system is not required for this process, since tissues isolated from PA- or plasminogen-deficient mice successfully neovascularize fibrin gels. By contrast, neovessel formation, in vitro and in vivo, is dependent on fibrinolytic, endothelial cell-derived matrix metalloproteinases (MMP). MMPs directly regulate this process as invasion-incompetent cells penetrate fibrin barriers when transfected with the most potent fibrinolytic metalloproteinase identified in endothelium, membrane type-1 MMP (MT1-MMP). Membrane display of MT1-MMP is required, as invasion-incompetent cells expressing a fibrinolytically active, transmembrane-deleted form of MT1-MMP remain noninvasive. These observations identify a PA-independent fibrinolytic pathway wherein tethered MMPs function as pericellular fibrinolysins during the neovascularization process.

Urokinase is required for the pulmonary inflammatory response to Cryptococcus neoformans. A murine transgenic model.

Urokinase (uPA) is hypothesized to provide proteolytic activity enabling inflammatory cells to traverse tissues during recruitment, and it is implicated as a cytokine modulator. Definitive evaluation of these hypotheses in vivo has previously been impossible because uPA could not completely and irreversibly be eliminated. This limitation has been overcome through the development of uPA-deficient transgenic mice (uPA-/-). Using these mice, we evaluated the importance of uPA in the pulmonary inflammatory response to Cryptococcus neoformans (strain 52D). C. neoformans was inoculated into uPA-/- and control mice (uPA+/+), and cell recruitment to the lungs was quantitated. The number of CFU in lung, spleen and brain was determined to assess clearance, and survival curves were generated. By day 21 after inoculation, uPA-/- mice had markedly fewer pulmonary inflammatory (CD45+), CD4+, and CD11b/CD18+ cells compared with uPA+/+ controls (P<0.0007); pulmonary CFUs in the uPA-/- mice continued to increase, whereas CFUs diminished in uPA+/+ mice(P<0.005). In survival studies, only 3/19 uPA+/+ mice died, whereas 15/19 uPA-/- mice died (p<0.001). We have demonstrated that uPA is required for a pulmonary inflammatory response to C. neoformans. Lack of uPA results in inadequate cellular recruitment, uncontrolled infection, and death.

The urokinase receptor (CD87) facilitates CD11b/CD18-mediated adhesion of human monocytes.

Urokinase receptors (uPAR; CD87) from complexes with complement receptor 3 (CR3) (CD11b/CD18), a beta2 integrin. In this study, we sought to determine if this association modulates the adhesive function of CR3. Both CR3 and uPAR concentrate at the ventral surface of fibrinogen-adherent human monocytes, and CR3-uPAR coupling increases substantially upon adhesion to fibrinogen. Pretreatment with anti-uPAR monoclonal antibody reduced adhesion to CR3 counterligands (fibrinogen and keyhole limpet hemocyanin) by 50%, but did not affect adhesion to fibronectin, a beta1 integrin counterligand. Antisense (AS) oligonucleotides were used to determine if selectively suppressing uPAR expression also modulates CR3 adhesive function. AS-uPAR oligo reduced CR3-dependent adhesion by 43+/-9% (P<0.01), but did not affect CR3-independent adhesion. To determine if the effects of uPAR are mediated through its ligand, monocytes were pre-treated with AS oligo to block uPA expression. Unlike the effects of blocking uPAR expression, AS-uPA oligo increased adhesion by 46% (P<0.005), and exogenous intact uPA, but not uPA fragments, reversed this effect. We conclude that complex formation with uPAR facilitates the adhesive functions of CR3. This function of uPAR is not dependent upon its occupancy with uPA, which negatively influences adhesion.

Endogenously produced urokinase amplifies tumor necrosis factor-alpha secretion by THP-1 mononuclear phagocytes.

This study examined the effects of endogenous urokinase (uPA) on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) secretion in THP-1 mononuclear phagocytes. Anti-uPA monoclonal antibody (mAb) suppressed LPS-driven TNF-alpha 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-alpha mRNA was suppressed in parallel with secreted TNF-alpha protein. TNF-alpha 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-alpha 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-alpha 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.

Function of the urokinase receptor (CD87) in neutrophil chemotaxis.

During recruitment, leukocytes respond to chemotaxins and traverse matrix barriers. Urokinase-type plasminogen activator (uPA), bound to its receptor (uPAR; CD87) facilitates plasmin formation, which promotes matrix proteolysis. Polymorphonuclear leukocytes (PMNs) are critical to the inflammatory response and express both uPA and CD87. To determine whether uPA and CD87 are required for PMN chemotaxis, PMNs were pretreated with an anti-CD87 monoclonal antibody (mAb), a neutralizing anti-uPA mAb, or uPA. PMN chemotaxis was profoundly suppressed by the anti-CD87 mAb but was unaffected by anti-uPA mAb or uPA. The role CD87 plays in chemotaxis may be related to its ability to associate with CR3. CD87/CR3 coupling can be disrupted by specific saccharides. The same saccharides that disrupt CD87/CR3 coupling (NADG, D-mannose, and mannoside) inhibit PMN chemotaxis. We conclude that CD87 plays a crucial role in PMN chemotaxis in vitro that is independent of uPA enzyme activity but may be related to the ability of CD87 to interact with CR3.

Enzyme-linked immunoabsorbent assay detection of a soluble form of urokinase plasminogen activator receptor in vivo.

The receptor for urokinase plasminogen activator (uPA-R, CD87) is a glycosylphosphatidylinositol (GPI)-anchored 50 to 65 kD glycoprotein that, by regulating membrane-associated plasmin activity, may facilitate the invasion of inflammatory and malignant cells. Certain other GPI-anchored glycoproteins are shed from the cell membrane and exist as soluble products in vitro and in vivo. To determine if uPA-R undergoes a similar phenomenon, we have developed a sensitive enzyme-linked immunoabsorbent assay (ELISA) (using a rabbit antiserum as both capture and detection reagents) to measure the quantity of soluble uPA-R (suPA-R) in tissue culture supernatants and biologic fluids. Using this ELISA, we have detected suPA-R in the culture supernatants of U-937 cells and human monocytes stimulated in vitro by certain soluble inflammatory mediators (Sitrin et al, Blood 84:1268, 1994; Mizukami et al., Clin Res 42:115A, 1994). To determine if suPA-R exists in vivo, we have screened the plasma of 20 normal volunteers (mean +/- SD, 3 +/- 3 ng/mL; median, 2 ng/mL; range, 1 to 11 ng/mL [serum values slightly higher]); the plasma of 13 ICU patients with clinical sepsis syndrome (mean +/- SD, 30 +/- 11 ng/mL; median, 11 ng/mL; range, 4 to 221 ng/mL); and the extravascular fluids (pleural, pericardial, and peritoneal) of 84 individuals with presumed inflammatory or malignant conditions (mean +/- SD, 21 +/- 39 ng/mL; median, 10 ng/mL; range, 2 to 253 ng/mL). Among the latter specimens, most were inflammatory exudates (only six were malignant by positive cytology) with the highest quantities of suPA-R associated with neutrophilic exudates. The solubility of suPA-R contained within these fluids was confirmed by reanalysis after ultracentrifugation to remove particulate material. When tested in a uPA ligand capture ELISA, representative specimens of extravascular body fluids and sepsis plasma contained suPA-R capable of binding uPA ligand (generally representing a small fraction of the immunoreactive material). We conclude from these data that suPA-R is immunologically detectable in vitro and in vivo with high concentrations of receptor found under conditions of inflammatory stimulation. The possibility of suPA-R's biologic activity is suggested by its partial retention of ligand binding capacity.

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.

Cytokine-specific regulation of urokinase receptor (CD87) expression by U937 mononuclear phagocytes.

Mononuclear phagocytes concentrate urokinase-type plasminogen activator (uPA) at the cell surface by expressing membrane uPA receptors (uPAR). This study examines the ability of exogenous cytokines to alter expression of membrane-associated uPA and uPAR in U937 mononuclear phagocytes. Cells were stimulated with recombinant interferon gamma (IFN gamma) or tumor necrosis factor alpha (TNF alpha), followed by immunolabeling for uPA or uPAR and flow cytometry. IFN gamma increased surface uPA 2.2-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no significant effect. Likewise, maximal uPA binding capacity was increased 2.8-fold by IFN gamma (P < .02), but was not affected by TNF alpha. In unstimulated cells, 50% of receptors were occupied by endogenously generated uPA, and this proportion was not affected by either cytokine. IFN gamma upregulated uPAR 2.1-fold relative to unstimulated controls (P < .001), whereas TNF alpha had no effect. In contrast to effects on surface protein, TNF alpha induced a substantial increase in uPAR mRNA, equaling the effect of IFN gamma. In addition, both cytokines doubled the intracellular uPAR pool (P < .01). By contrast, TNF alpha induced a 2.5-fold increase in the level of uPAR protein released into conditioned medium (compared with unstimulated cells), whereas IFN gamma had no effect. These results indicate that uPAR expression is regulated in a cytokine-specific fashion. Some stimuli, such as TNF alpha, may increase uPAR synthetic activity without a corresponding change in membrane expression, because of enhanced release of uPAR from the cell. Cytokine-specific modulation of uPAR may be important in regulating the function of mononuclear phagocytes in inflammation and tissue repair.

Experimental murine pulmonary cryptococcosis. Differences in pulmonary inflammation and lymphocyte recruitment induced by two encapsulated strains of Cryptococcus neoformans.

BACKGROUND: Cryptococcus neoformans, the most common cause of lethal mycosis in AIDS, usually causes only subclinical pneumonitis in normal hosts. However, cryptococcosis can induce various pulmonary inflammatory reactions, and pulmonary cellular immunity is postulated to prevent dissemination. We hypothesized that cryptococcal strains possess different capacities to induce recruitment to the lungs of inflammatory cells, especially lymphocytes, which are necessary for cryptococcal clearance. EXPERIMENTAL DESIGN: We examined the pulmonary response of CBA/J mice to intratracheal inoculation with C. neoformans of either of two strains: 52D (ATCC 24067), which rarely kills immunocompetent mice; and 145A (ATCC 62070), which is uniformly fatal. From 2-42 days after inoculation, lungs were either examined grossly and microscopically or were enzymatically digested and inflammatory cells counted and analyzed by flow cytometry. At 42 days, organism burden in lung and brain was quantified by colony-forming unit assay. RESULTS: Pulmonary inflammation differed greatly between the two strains. Strain 52D induced dense perivascular and alveolar inflammation; infection progressed to day 21 and then waned. In contrast, strain 145A induced delayed, meager lymphocytic infiltration and slight alveolitis; organisms grew progressively. Recovery of inflammatory cells increased by day 13 with strain 52D, but not until day 31 with strain 145A. Although all lymphocyte subsets were greater in 52D infection, the disparity was greatest for CD4+ T cells. Nevertheless, lymphocytes from paratracheal nodes of infected mice proliferated in vitro to heat-killed cryptococci, indicating immune recognition of both strains. At day 42, strain 52D lightly infected lungs but not brain, whereas strain 145A heavily infected lungs and brain. CONCLUSIONS; Cryptococcal strains differ in their capacity to induce pulmonary cellular inflammation, especially CD4+ T cell recruitment. Our results suggest that strain-specific difference in the organism's ability to induce (or evade) pulmonary inflammation contributes to the outcome of infection.

The urokinase receptor is required for human monocyte chemotaxis in vitro.

Mononuclear phagocytes (Mphi) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which Mphi can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in Mphi chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or F[ab']2) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M phi chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during M phi migration.

Giant splenomegaly and refractory hypercalcemia due to extrapulmonary sarcoidosis. Successful treatment by splenectomy.

Sarcoidosis is a granulomatous disease of unknown origin with a variable clinical presentation. The presenting complaint is usually referable to the lung. We describe an unusual presentation of sarcoidosis in a young black man who received medical attention for evaluation of pancytopenia, giant splenomegaly, and marked, refractory hypercalcemia. After extensive evaluation, including exploratory laparotomy, he was found to have sarcoidosis, with extensive involvement of his spleen, liver, and abdominal lymph nodes. Pulmonary involvement was notably absent, with no suggestive findings radiographically on gallium citrate Ga 67 scanning or on bronchoscopy with transbronchial biopsy. This patient underwent splenectomy and, following removal of the massive splenic granuloma burden, the hypercalcemia resolved completely with no other therapy.

Immunology of the aging lung.

The aging process is associated with multiple deficits in pulmonary immune function. Defense of the airway is impaired in the elderly by decreased mucociliary clearance, alteration in respiratory mechanics and, in some cases, concomitant illnesses that predispose to aspiration. Alveolar defenses can be divided into resident defense mechanisms, inflammatory responses, and specific immune responses. Resident defenses such as macrophage phagocytosis and chemotaxis, although largely intact, may have subtle defects under specific conditions. Inflammatory responses may also be diminished, as evidenced by decreased neutrophil-mediated killing and chemotaxis. Specific immune responses appear to be the most vulnerable to age-associated impairment. Although antigen presentation is well preserved during aging, accessory cell cytokine production may be decreased. T-lymphocyte proliferative responses are markedly decreased, as is the production of and response to intercellular mediators. Age-associated alterations in T cell subpopulations may result in imperfect T cell-B cell interactions leading to expression of abnormal immunoglobulins. These many interacting and compounding impairments may explain the increased susceptibility of the elderly to pulmonary infection and autoimmune diseases.

Monocyte 1 alpha-hydroxylase regulation: induction by inflammatory cytokines and suppression by dexamethasone and uremia toxin.

Alveolar macrophages acquire 1 alpha-hydroxylase activity in inflammation, and thereby metabolize 25 hydroxyvitamin D3 (25 D3) to the active metabolite, 1 alpha,25-dihydroxyvitamin D3 (1,25 D3, calcitriol). Calcitriol is a potent differentiation agent that modulates mononuclear phagocyte activation and effector functions. The mediators that induce macrophage 1 alpha-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 1 alpha-hydroxylase activity, blood monocytes were cultured in the presence and absence of human recombinant tumor necrosis factor alpha (TNF-alpha), interferon-gamma (IFN-gamma), 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).

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, PAI-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-alpha and interferon-gamma 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.

Urokinase expression in mononuclear phagocytes: cytokine-specific modulation by interferon-gamma and tumor necrosis factor-alpha.

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 determine how recombinant interferon-gamma (IFN) and tumor necrosis factor-alpha (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 cytokine-rich inflammatory foci.