12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G.

Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast-like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o-tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is characteristic of monocytes, these data suggest that neutrophilic proteinases may be produced by normal monocytes during the early stages of their differentiation.     

A fast-acting elastase inhibitor in human monocytes

A proteinase inhibitor active against neutrophil and pancreatic elastase was detected in extracts of cultured human monocytes and the human monocyte-like cell line U937. This component forms a covalent complex with the active site of elastase; the complex is stable in boiling sodium dodecyl sulfate solution, and is susceptible to nucleophilic cleavage. The activity of the elastase inhibitor is not detected in extracts of freshly isolated monocytes, but becomes detectable when the monocytes are allowed to mature in culture, with maximum levels occurring at 5-7 d. The monocyte inhibitor is fast-acting; its reaction with 125I-labeled elastase is complete in less than 1 min at 37 degrees C. Analysis by electrophoresis and studies using a heteroantiserum to alpha 1-proteinase inhibitor demonstrated that the elastase inhibitor of monocytes/U937 cells is not identical to alpha 1-proteinase inhibitor, the major elastase inhibitor of blood plasma. The extent of conversion of 125I-elastase to the 125I-elastase-inhibitor complex is proportional to the amount of U937 extract or cultured monocyte extract, indicating that this reaction can serve to quantify the elastase inhibitor. The elastase inhibitor is an abundant component in mature monocytes, with greater than or equal to 1.5 X 10(6) molecules/cell (greater than or equal to 12 micrograms per 10(8) cells, greater than 0.1% of total cell protein). Its mol wt is estimated at 50,000. Thus, the monocyte inhibitor should be classified as a putative regulator of neutrophil (and monocyte) elastase activity at inflammatory sites. This designation is based on the properties of the molecule, including its high concentration in maturing monocytes, its affinity for elastase, and its fast reaction with this enzyme.     

Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G

A profound decrease in activities of the two lysosomal serine proteinases, elastase, and cathepsin G, was found in neutrophils of four independent beige mutants. Elastase and cathepsin G activities were assayed with the specific synthetic substrates MeO-Suc-Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively. The defect is intrinsic to cells of beige mice, since transplantation of bone marrow from normal to mutant mice restored normal proteinase activity, and normal mice transplanted with beige marrow produced neutrophils with a deficiency of proteinase activity. The loss of elastase and cathepsin G activity was confirmed by separation of [3H]diisopropylfluorophosphate-labeled proteins on denaturing gels, which also revealed that other serine proteinases are at normal levels in beige neutrophil extracts. The deficiency of lysosomal proteinase activity appears specific, in that four other common neutrophil lysosomal enzymes, plus the spectrum of major neutrophil proteins are not affected by the beige mutation. The deficiency of proteinase activity is likely not the primary genetic alteration of the beige mutation, since more than one proteinase is affected, and heterozygous F1 mice have normal rather than intermediate levels of both proteinases. The lowered proteinase activity may contribute to the high susceptibility of beige mice and Chediak-Higashi patients to infection.     

Identification of Human Neutrophil-derived Cathepsin G and Azurocidin/CAP37 as Chemoattractants for Mononuclear Cells and Neutrophils

Macrophage infiltration into inflammatory sites is generally preceded by neutrophils. This suggests neutrophils may be the source of chemotactic factors for monocytes. To identify these putative monocyte attractants, we have systematically prepared neutrophil granules, lysed them, and sequentially purified the released proteins by several reverse phase chromatography procedures. Assays for monocyte chemotactic activity of the chromatography fractions yielded a major peak of activity associated with a protein of 30 kD, according to SDS-PAGE analysis. NH₂-terminal sequence of the protein revealed this to be identical to cathepsin G. The monocyte chemotactic activity of human cathepsin G was dose dependent with optimal concentration at 0.5–1 μg/ml. Cathepsin G is chemotactic rather than chemokinetic for monocytes, as demonstrated by checkerboard analysis. Cathepsin G–induced monocyte chemotaxis is partially pertussis toxin sensitive implying the involvement of a G protein–coupled receptor. Enzymatic activity of cathepsin G is associated with its monocyte chemotactic activity, since DFP- or PMSF-inactivated cathepsin G no longer induced monocyte migration. The chemotactic activity of cathepsin G can also be completely blocked by α₁ antichymotrypsin, a specific inhibitor of chymotrypsin-like proteinases present in human plasma. In addition, cathepsin G is also a potent chemoattractant for neutrophils and a chemokinetic stimulant for T cells. In the course of pursuing these in vitro studies, we established that the T cell chemoattractant, azurocidin/CAP37 from human neutrophil granules, at doses of 0.05 to 5 μg/ml, was chemotactic for monocytes and neutrophils. As predicted from the in vitro chemotactic activity, subcutaneous injection of cathepsin G into BALB/c mice led to infiltration of both mononuclear cells and neutrophils. Thus, the transition of inflammatory exudate from neutrophil to mononuclear cells can be mediated, at least in part, by extracellular release of neutrophil granule proteins such as cathepsin G and azurocidin/CAP37.     

Elastase and cathepsin G activities are present in immature bone marrow neutrophils and absent in late marrow and circulating neutrophils of beige (Chediak-Higashi) mice

Elicited peritoneal neutrophils of beige (Chediak-Higashi) mice essentially lack activities of the neutral serine proteinases elastase and cathepsin G, which may explain the increased susceptibility to infection of beige mice and Chediak-Higashi patients. We have examined neutrophils of beige mice at earlier points in their development to determine if the proteinase genes are never expressed or whether they are expressed and then lost during neutrophil maturation. Surprisingly, bone marrow of beige mice had significant elastase and cathepsin G activity (approximately 60% of normal). The results of several experiments indicate that neutrophils were the sole source of elastase and cathepsin G in bone marrow. Neutral proteinase activity was readily demonstrable by histochemical procedures in beige marrow neutrophil precursors up to and including the metamyelocyte stage. However, mature neutrophils of beige marrow had greatly decreased activity. Also mature neutrophils (PMNs) of the peripheral circulation, like peritoneal neutrophils, had very low elastase and cathepsin C activities. Thus we conclude that beige neutrophil precursors express neutral proteinase activity, which is largely and irreversibly depleted by the time they fully mature in marrow.     

Characterization of infections of human leukocytes by non-polio enteroviruses

To elucidate the detailed susceptibilities of leukocytes to clinically important non-polio enteroviruses (EVs), primary monocytes and various human leukocyte cell lines were infected with coxsackievirus A24 (CVA24), coxsackievirus B3 (CVB3), and enterovirus 70 (EV70). The permissiveness was then assessed by determining virus replication and resultant cytopathic effects. Different EVs varied markedly in their ability to infect leukocyte cell lines. CVB3 replicated effectively in leukocytes of B-cell, T-cell, and monocyte origin, CVA24 in leukocytes of B-cell and monocyte origin, and EV70 in leukocytes of monocyte origin. Primary monocytes, as well as monocyte-derived U-937 cells, were permissive to all three EVs. We observed a positive correlation between cytotoxicity and active virus replication, except in CVB3-infected monocytes. U-937 cells efficiently generated CVB3 progeny virus without severe cellular damage, including cell death. Moreover, infectivity on leukocytes was not absolutely associated with the availability of viral receptors. These findings suggest that the susceptibility of human leukocytes to non-polio EVs may be responsible for virus transport during the viremic phase, particularly to secondary target organs, and that active replication of CVB3 in all human leukocyte lineages leads to greater dissemination, in agreement with the ability of CVB to cause systemic diseases.     

Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils

A fast-acting inhibitor of serine elastase has been detected at high levels in human neutrophils, fresh monocytes, matured monocytes, and macrophages. The elastase inhibitor was isolated from large scale cultures of the monocyte-like cell line U937 by DNase chromatography, disulfide exchange, Phenyl-Sepharose, Red A-agarose, and DEAE HPLC chromatography with an average yield of 480 micrograms from 1.8 x 10(10) cells. The isolated polypeptide was verified as elastase inhibitor by its ability to (a) form a covalent complex with elastase; and (b) inhibit the elastinolytic activity of elastase. The purified elastase inhibitor molecule is unique, i.e., physiochemical and/or functional properties distinguish it from all other serine proteinase inhibitors. Treatment with iodoacetamide abrogates the ability of the molecule to form a complex with elastase, thereby providing evidence for the presence of an essential cysteine residue. Based on functional criteria, this elastase inhibitor has been grouped with the proteinase inhibitors of the serpin superfamily. The purified elastase inhibitor is a single polypeptide of Mr approximately 42,000. The NH2 terminus appears to be blocked. Compositional analyses indicates five cysteine residues per molecule of approximately 360 amino acid residues. Negligible levels of carbohydrate were detected on gas-liquid chromatography. This finding and the insensitivity of the molecule to peptide N-glycosidase F treatment strongly indicate that the elastase inhibitor is a nonglycosylated protein.     

Modulation of monocyte-endothelial cell interactions by platelet microparticles.

Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.     

Elastase regulates the synthesis of its inhibitor, alpha 1-proteinase inhibitor, and exaggerates the defect in homozygous PiZZ alpha 1 PI deficiency.

The net balance of neutrophil elastase, an enzyme that degrades many components of the extracellular matrix, and its inhibitor, alpha-1-proteinase inhibitor (alpha 1 PI), is thought to be a critical determinant in the development of destructive lung disease, especially in individuals with homozygous alpha 1 PI deficiency. Synthesis and secretion of alpha 1 PI has been recently demonstrated in cells of mononuclear phagocyte lineage, including peripheral blood monocytes and tissue macrophages. In this study we show that alpha 1 PI gene expression in human monocytes and bronchoalveolar macrophages is affected by a novel mechanism, whereby elastase directly regulates the synthesis of its inhibitor. In nanomolar concentrations, neutrophil or pancreatic elastase mediates a dose- and time-dependent increase in steady state levels of alpha 1 PI mRNA and in the rate of synthesis of alpha 1 PI in human monocytes and bronchoalveolar macrophages. Antisera to neutrophil elastase or pretreatment of elastase with the serine proteinase inhibitor diisopropylfluorophosphate abrogates the effect of elastase on alpha 1 PI expression. Elastase also stimulates the synthesis of alpha 1 PI in monocytes from homozygous PiZZ alpha 1 PI-deficient individuals, but has no effect on the rate of secretion; hence, the enzyme mediates an effect on alpha 1 PI that increases the intracellular accumulation of inhibitor and exaggerates the intrinsic defect in secretion of alpha 1 PI that characterizes the homozygous PiZZ alpha 1 PI deficiency.     

Vα24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages

Tumor infiltration with Vα24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d⁺ cells, the majority of human tumors are CD1d^–. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68⁺ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.     

Transient elevation of neutrophil proteinases in induced sputum during COPD exacerbation

Objective. Patients with chronic obstructive pulmonary disease (COPD) are prone to acute exacerbations associated with increased morbidity and mortality. One potential group of enzymes causing tissue destruction in this disease includes neutrophil proteinase elastase (NE), collagenase‐2 (matrix metalloproteinase‐8 (MMP‐8)) and gelatinase B (MMP‐9). We investigated the activity of NE and the levels of MMP‐8 and MMP‐9 in a longitudinal setting at and after COPD exacerbation using a non‐invasive technique, i.e. induced sputum, to ascertain whether these proteinases play a role in COPD exacerbation. Material and methods. The study included healthy non‐smokers (n = 32), healthy smokers (n = 28), patients with stable COPD (n = 15), COPD patients with acute exacerbations (exa) (n = 10) and their recovery (n = 8) after 4 weeks. NE activity by synthetic peptide substrate and spectrophotometry, MMP‐8 levels by immunofluorometry and MMP‐9 levels by ELISA were analysed from induced sputum supernatants. Results. NE activity and the level of MMP‐8 increased highly significantly in patients with COPD exacerbation compared to stable COPD and controls (NE: p = 0.001 and p<0.0001; MMP‐8: p<0.001 and p<0.0001). Paired samples showed a decrease of these proteinases during the recovery period after exacerbation (p = 0.03, p = 0.04). The proteinase levels correlated not only with the percentage and number of neutrophils but also with the lung function parameters (FEV1/FVC and diffusion capacity). Conclusions. COPD exacerbations are associated with neutrophil recruitment into the airways but also transient activation and/or elevation of tissue destruction proteinases, such as NE and MMP‐8, which can be detected from the induced sputum supernatants of these COPD patients.     

Constitutive secretion of cystatin C (gamma-trace) by monocytes and macrophages and its downregulation after stimulation

Cystatin C (gamma-trace) was found to be a constitutively secreted protein of isolated human monocytes and mouse peritoneal macrophages, as well as the histiocytic lymphoma cell lines U937, P388D.1, and J774. This proteinase inhibitor is not uniquely secreted by monocytes/macrophages, but was also identified in the conditioned media from several primary cells, including brain cells, and diverse established cell lines. In vitro treatment of resident mouse peritoneal macrophages with either LPS or IFN-gamma caused a downregulation in cystatin C secretion. Elaboration of this protein was also diminished by macrophages that had been stimulated by thioglycollate in vivo, and treatment of these cells with LPS led to further decline. It is suggested that, under some inflammatory conditions, downregulation of cystatin C may contribute to tissue pathology.     

Proteolytic destruction of functional proteins by phagocytes in human peritonitis

BACKGROUND: Besides phagocyte-derived oxidative autoaggression, proteolytic destruction of functional proteins in the peritoneal cavity may also be involved in the pathomechanism of secondary peritonitis. To evaluate the pattern of proteolysis, 43 patients undergoing initial operation for acute peritonitis (n = 30) or scheduled abdominal lavages (Etappenlavage) for resolution of persistent peritonitis (n = 13) and 16 surgical patients with abdominal exudation without peritonitis were enrolled in our study. MATERIALS AND METHODS: Thirty blood samples and purulent exudates were taken simultaneously in each peritonitis group at the surgical interventions. Sixteen clear exudates were obtained from patients with post-operative non-infectious irritations. The following parameters were measured: (a) elastase (from neutrophils) and cathepsin B (from monocytes/macrophages); (b) alpha1-proteinase inhibitor (alpha1PI) and overall cysteine proteinase inhibitor capacity; and (c) opsonic activity and degradation products of fibrinogen, complement C3 and immunoglobulin IgG. RESULTS: Circulating levels of phagocyte proteinases and of alpha1PI were significantly elevated, whereas antigen concentrations and opsonic activity of C3 and IgG were slightly reduced in peritonitis patients compared to healthy volunteers. No degradation products were detectable in patients' blood. Discharge of phagocyte proteinases was even more pronounced in both types of peritonitis exudates. Although most of the elastase was complexed with alpha1PI, active elastase and its specific fibrinogen split product was found along with significantly reduced inhibitory capacity for elastase and cysteine proteinases. Local opsonic activity was dramatically diminished because of proteolytic degradation of C3 and IgG. Despite some phagocyte proteinase release, no destruction of functional proteins was seen in clear exudates. CONCLUSIONS: Higher values of extracellularly released phagocyte proteinases concomitant with lower opsonin activity in exudates from patients with persistent peritonitis can be taken as a further hint of the involvement of local proteolysis-induced pathomechanisms in the development of lethal multiple organ failure, which occurred more frequently in patients with persistent peritonitis (54%) than in those with acute peritonitis (27%).     

Human Alveolar Macrophage-derived Chemotactic Factor for Neutrophils: STIMULI AND PARTIAL CHARACTERIZATION

The presence of neutrophils within the lung is a characteristic feature of a variety of lung diseases. To evaluate the potential role of alveolar macrophages in modulating the migration of neutrophils to the lung, normal human alveolar macrophages obtained from volunteers by bronchopulmonary lavage, were exposed for various periods of time in vitro to heat-killed microorganisms, and noninfectious particulates, immune complexes, and the macrophage supernates were evaluated for chemotactic activity. The microorganisms, noninfectious particulates, and immune complexes were chosen as stimuli for alveolar macrophages because these stimuli are representative of a spectrum of pathogenic agents that cause neutrophil accumulation in the lower respiratory tract. After incubation with each of these stimuli, alveolar macrophages released low molecular weight (400-600) chemotactic factor(s) (alveolar macrophage-derived chemotactic factor[s] [AMCF]) with relatively more activity for neutrophils than monocytes or eosinophils. Checker-board analysis of the AMCF revealed that the factor was primarily chemotactic and not chemokinetic for neutrophils. The selectivity for neutrophils vs. monocytes could not be explained by a selective deactivation of monocytes, because the AMCF was more potent in deactivating neutrophils than monocytes. Partial characterization of AMCF demonstrated it was heterogeneous with the following features: (a) stable to heating at 56 and 100°C for 30 min; (b) stable over a pH range of 1.0 to 12.0 for 60 min; (c) stable after exposure to trypsin, papain, chymotrypsin, collagenase, and elastase; (d) partially inhibited by serum chemotactic factor inhibitor(s); (e) two major isoelectric points (pI 7.6 and 5.2); and (f) partially extractable into ethyl acetate, ether, and hexane. Although AMCF was, at least, partially lipid in nature, it did not appear to be similar to previously described lipid chemotactic factors (e.g., hydroxy-derivatives of 5,8,10,14-eicosatetraenoic acid); analysis by gas chromatography-mass spectrophotometry of AMCF extracted into ethyl acetate did not reveal the presence of 5,8,10,14-eicosatetraenoic acid. The macrophage supernates containing the AMCF also stimulated normal human neutrophils to release lysozyme and lactoferrin but not lactate dehydrogenase. These studies suggest that a wide variety of potentially pathogenic stimuli induce normal alveolar macrophages to generate a low molecular weight chemotactic factor(s) that preferentially attracts neutrophils. Because alveolar macrophages are normal residents of alveoli, it is likely that by releasing this factor(s) macrophages play a significant role in amplifying the inflammatory processes seen in many acute and chronic lung diseases.     

Murine gamma interferon activates the release of a macrophage-derived Ia-inducing factor that transfers Ia inductive capacity

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.     

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN-gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN-beta competed only weakly. Monoclonal antibodies against IFN-gamma either inhibited or enhanced MAF activity by blocking or increasing IFN-gamma binding to macrophages, respectively. These results indicate that IFN-gamma reacts with a receptor on macrophage in a specific and saturable manner and this interaction initiates macrophage activation.     

Measurement of elastase and cysteine proteinases in synovial fluid of patients with rheumatoid arthritis, sero-negative spondylarthropathies, and osteoarthritis.

Synovial fluid samples were collected from 45 patients with rheumatoid arthritis, spondylarthropathy, or osteoarthritis, to study their content of elastase (EC 3.4.21.37) and of cysteine proteinases (EC 3.4.22.1, 3.4.22.15). We measured both elastase complexed with alpha 1-proteinase inhibitor and elastase activity toward the substrate L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide. Cysteine proteinase activities were measured with the substrates N-benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and Z-Arg-Arg-AMC and the inhibitor E-64 [L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino)-butane]. In all these enzyme assays, higher median values were obtained in inflammatory arthropathies than in osteoarthritis. The concentration of the elastase-alpha 1-proteinase inhibitor complex and of elastase and cysteine proteinase activities were statistically higher in patients with rheumatoid arthritis than in patients with osteoarthritis. The difference in results between patients with spondylarthropathy and patients with osteoarthritis was statistically significant only for the elastase-alpha 1-proteinase inhibitor complex. The median values of the complex and of both enzyme activities were higher in patients with rheumatoid arthritis than in patients with spondylarthropathy; however, the difference was statistically significant only for the cysteine proteinase activity measured with Z-Arg-Arg-AMC substrate. These results suggest that both elastase and cysteine proteinases, which are increased in patients with inflammatory arthritis, are involved in cartilage degradation in these arthropathies.     

THE ORIGIN AND KINETICS OF MONONUCLEAR PHAGOCYTES

The origin and turnover of efferent populations of mouse mononuclear phagocytes has been described. Mononuclear phagocytes were defined as mononuclear cells which are able to adhere to glass and phagocytize. In vitro labeling studies with thymidine-³H showed that monocytes in the peripheral blood and peritoneal macrophages do not multiply and can be considered end cells in a normal, steady state situation. However, the mononuclear phagocytes of the bone marrow appear to be rapidly dividing cells. This conclusion was supported by in vivo labeling experiments. A peak of labeled mononuclear phagocytes of the bone marrow was found 24 hr after a pulse of thymidine-³H. This was followed, 24 hr later, by a peak of labeled monocytes in the peripheral blood. From these experiments it was concluded that the rapidly dividing mononuclear phagocytes of the bone marrow, called promonocytes, are the progenitor cells of the monocytes. Labeling studies after splenectomy and after X-irradiation excluded other organs as a major source of the monocytes. Peak labeling of both the blood monocyte and peritoneal macrophages occurred at the same time. A rapid entry of monocytes from the blood into the peritoneal cavity was observed, after a sterile inflammation was evoked by an injection of newborn calf serum. These data have led to the conclusion that monocytes give rise to peritoneal macrophages. No indications have been obtained that mononuclear phagocytes originate from lymphocytes. In the normal steady state the monocytes leave the circulation by a random process, with a half-time of 22 hr. The average blood transit time of the monocytes has been calculated to be 32 hr. The turnover rate of peritoneal macrophages was low and estimated at about 0.1% per hour. On the basis of these studies the life history of mouse mononuclear phagocytes was formulated to be: promonocytes in the bone marrow, → monocytes in the peripheral blood, → macrophages in the tissue.