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C 2003) Inflammation, Vol. 27, No. 6, December 2003 (°

Modulation of Macrophage Activity by Proteolytic Enzymes. Differential Regulation of IL-6 and Reactive Oxygen Intermediates (ROIs) Synthesis As a Possible Homeostatic Mechanism in the Control of Inflammation K. Bryniarski,1 K. Maresz,1 M. Szczepanik,2 M. Ptak,1 and W. Ptak1,3

Abstract—Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-α production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function. KEY WORDS: macrophages; proteolytic enzymes; cell surface antigens; IL-6; oxygen intermediates.

INTRODUCTION

of increased vasopermeability caused by local mediators liberated by damaged tissue, granulocytes actively extravasate and accumulate at the site of injury within 1 h attracted by leucotactic factors various origin (lymphocyte products, bacterial metabolites, fibrin split products). Within several hours monocytes and macrophages allured by similar mechanism arrive at the inflammatory site and later during healing or in chronic inflammation become predominating cells. Production of reactive oxygen intermediates (ROI) and discharge of enzymes by phagocytes results in damage to neighboring cells and to phagocytic cells themselves. Bacterial products, particularly proteolytic enzymes, can contribute to tissue damage (1–7). Normally inflammatory reaction is under strict control

Inflammation arises as a sequel of a complex series of events that follow tissue damage caused by mechanical, thermal, or chemical trauma, or by microbial products. It is an essentially protective and restorative reaction which, at least in its first phase, has stereotypic course. Because 1 Department

of Immunology, Medical College of Jagiellonian University, Cracow, Poland. 2 Department of Human Developmental Biology, Medical College of Jagiellonian University, Cracow, Poland. 3 To whom correspondence should be addressed at Department of Immunology, CMUJ, ul. Czysta 18, 31-121 Krak´ow, Poland. E-mail:[email protected]

333 0360-3997/03/1200-0333/0

° C

2003 Plenum Publishing Corporation

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334 of several regulatory mechanisms (2), and if they fail or are aberrant, consequences may be deleterious. The biological role of macrophages in the development of inflammation is not necessarily identical during the whole course of reaction. We reasoned that the local environment of developed inflammatory focus rich in proteolytic enzymes can direct later arriving macrophages into alternative pathways rather than encourage them to perform solely effector functions. Our paper shows that this indeed may be the case, since macrophages treated in vitro with proteolytic enzymes severely limited the production of ROIs and significantly upregulated the amount of produced IL-6. MATERIALS AND METHODS Animals Inbred CBA/J male mice from our own breeding unit weighing 22–25 g were used throughout these experiments. Reagents The following reagents were used collagenase type IA, trypsin from bovine pancreas (T1426), lucigenin (bis-N -methylacridinum nitrate), zymosan A, o-phenylenediamine, hydrogen peroxide, MTT (3,4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, isopropanol, recombinant murine TNF-α, (Sigma, St. Louis, MO); ethylenediamine-tetra-acetic acid (EDTA; BDH, Poole, UK); RPMI 1640, fetal calf serum (FCS; Gibco Life Technologies, Grand Island, NY); trypsin, (Difco Laboratories, Detroit, MI); recombinant mouse IL-6 (PeproTech, Rocky Hill, NY), recombinant murine IL-12 (Genzyme, Cambridge, MA); monoclonal rat antimouse IL-6 (MP5-20F3), biotinylated monoclonal rat anti-mouse IL-6 (MP5-32C11), monoclonal rat antimouse IL-12 (C15.6), monoclonal rat anti-mouse TNFα (G281-2626), biotinylated monoclonal rat anti-mouse TNF-α (MP6-XT3), FITC conjugated rat anti-mouse Mac-3 and anti-CD11b mAbs, PE-conjugated anti-mouse CD32/16, anti-CD23, anti-CD95L, anti-CD14, IL-10 OptEIATM ELISA set (all from BD PharMingen, San Diego, CA); biotinylated monoclonal rat anti-mouse IL12 (C17.8) (Endogen, Woburn, MA); FITC-conjugated anti-F4/80 (Serotec, Oxford, UK); anti CD32/16 (2.4G2) was a gift from Dr K. Eichmann, Max Planck Institute of Immunobiology (Freiburg, FRG); Rat IgG2a (Caltag Lab., Burlingame, CA); paraffin oil Markol 52 (Exxon

Bryniarski, Maresz, Szczepanik, Ptak, and Ptak Corp., New York, NY); heparin (Polfa, Warsaw, Poland); horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA); pyrogen-free distilled water (PolishAmerican Institute of Paediatrics, CM UJ (Cracow, PL); granulated milk (Marvel, Chivers Ltd, Coolock, Ireland). For cell cultures Nunc lab ware (Roskilde, DK) were used throughout. Isolation of Macrophages Peritoneal macrophages (Mf) were collected from peritoneal cavity of naive mice (resident Mf, R-Mf) or from mice which four days before cell harvesting were injected i.p. with 1 mL of Markol 52 mineral oil (Oil-Mf). Three to six mice were used per group. Mφgere washed out with 5 mL of phosphate buffered saline (PBS) containing 5-U heparin per mL. Oil-induced peritoneal cells contained over 90% macrophages (FcR-and esterase-positive cells) and were not purified further. R-Mf with Mf content below 60% were purified from granulocytes by discontinuous gradient centrifugation (30 min, 700 × g, 20◦ C) on the Ficoll–Paque medium (ρ = 1.077g/cm3 ) and collected from the upper interface. This fraction contained over 90% Mf and some lymphocytes. Then aliquots of cells were treated for 30 min with serial fivefold dilution of proteolytic enzymes (starting with 40 µM concentrations in PBS adjusted for trypsin to pH 7.6 and collagenase to pH 7.0). After the enzyme treatment cells were washed twice in excess PBS containing 20% FCS and resuspended in RPMI 1640 medium supplemented with 5 or 10% FCS. The cell viability was assessed by trypan blue exclusion test and MTT test. For further tests cells were allowed to adhere for 2 h at 37◦ C in appropriate labware and then supernatants were removed and cultures supplemented with fresh medium. Tests were performed at 24 and sometimes at 48 h culture. We found that the enzyme treatment does not influence the long-term in vitro survival of Mf. Thus at the end of 24 or 48 h cultures cells were detached from wells at 4◦ C with 0.02% EDTA solution in PBS and cell count and final viability was estimated, by MTT and trypan blue exclusion tests. Estimation of Cell Viability The percentage of live cells was determined by standard trypan blue exclusion test and by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye metabolism method in live cells mitochondria (8). To 2 × 106 cells in 1.5 mL RPMI 1640 medium 100 µL of

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Modulation of Macrophage Activity by Proteases Flow Cytometry

Fig. 1. Viability of macrophages treated with proteolytic enzymes. Oil induced macrophages were treated with serial fivefold dilution of collagenase or trypsin (40, 8, 1.6, 0.3, 0.06 µM) for 30 min at 37◦ C or left untreated (1 × 107 cells/1 mL of enzyme solution). Two ×106 Oil-Mf in 1.5 RPMI 1640 medium were placed in triplicate in 12-well plastic trays and then MTT was added. Color reaction was measured at 570 nm (for details see Materials and Methods). Results are expressed as percent viability of treated cells ±SD against control cells = 100% viability. Only results with high (40 µM), moderate (1.6 µM), and low (0.06 µM) concentrations of enzymes are shown. The range of simultaneous estimation of viability by trypan blue exclusion test are also shown between two parallel lines. Identical results as with Oil-Mf were obtained with resident peritoneal macrophages (results not shown). Macrophages used in tests after overnight culture show viability ranging between 90 and 100% (not shown).

0.5% solution of MTT was added and left for 3.5 h. The plate was then frozen and thawed twice and treated with 1.9 mL of isopropanol solution with HCL (100 mL of isopropanol + 4 mL 1M HCL). It was then vigorously shaken to completely break up the cells. The extracted supernatants were spun down and absorbance was determined at 570 nm. The results obtained with control nontreated cells were taken as 100% (Fig. 1).

Lucigenin-Dependent Chemiluminescence Mf were cultured overnight at the concentration of 5 × 105 viable cells/well in 0.2 mL RPMI 1640 medium supplemented with 10% FCS in 96-well flat bottom dark plates (Nunc, Roskilde, Denmark). After that 10 µM lucigenin was added and cells were incubated for 15 min at 37◦ C in a dark adaptation (9). Then some groups of Mf were stimulated by zymosan opsonized with mouse serum (in 10 particles per cell ratio) and plates were immediately transferred to a Lucy 1 luminometer (Anthos, Salzburg, Austria) and the photon emission was measured for 75–100 min. Each experiment was run in duplicate.

Two to 3 × 107 Mf non-treated or pretreated with proteolytic enzymes were cultured overnight in Petri dishes (10 cm diameter) in RPMI 1640 medium containing 10% FCS since it was found in preliminary experiments that Mf labelled with fluorochrome-conjugated antibodies immediately after enzyme treatment show a significant non-specific fluorescence. Then adhering cells were detached by treatment with 0.02% EDTA in PBS at 4◦ C and in all groups (except CD32/16 and CD23) Fcγ receptors were blocked with 2.4 G2 mAb, rat IgG2a, and mouse Ig. Subsequently Mf were stained with appropriately diluted FITC-conjugated anti- F4/80, anti-Mac-3, or antiCD11b mAbs, or with PE-conjugated anti-CD32/16, antiCD23, anti-CD95L, anti-CD14. Cell surface immunotypic analysis of Mf was performed by cytofluorography using Ortho Cytoronabsolute flow cytometer collecting 1 × 104 cells (Ortho Diagnostic Systems, Raritan, NJ). Immunocount II software was used for data acquisition and analysis.

Cytokine Immunoassay Non-treated or enzyme-treated peritoneal macrophages were cultured in 24-flat bottom plates at a concentration of 5 × 105 /mL in RPMI 1640 medium supplemented with 5% FCS. Supernatants were collected after 48 h and frozen in −80◦ C until further use. Concentrations of IL-6, IL-12, and TNF-α in culture supernatants were measured in capture ELISA test, using Corning Easy Wash plates (Corning, Corning, NY). Recombinant murine cytokines were used as standards. To each well a suitably diluted antibody was added (50 µL, or in the case of IL-10, 100 µL). After an overnight incubation wells were washed twice with PBS containing 0.1% Tween 20 and then blocked with 200 µL of 3% granulated milk in PBS. Plates were thoroughly washed and after the addition to each well of 100 µL of appropriately diluted standards or tested supernatants incubated overnight at 4◦ C. After several washes 100 µL of biotinylated antibody was added to each well and plates were incubated for 1 h at 37◦ C and again thoroughly washed. Then after addition of 100 µL of streptavidin-HRP and 100 µL o-phenylenediamine+ H2 O2 to each well plates were incubated for 30 min at room temperature and reaction was stopped with 3 M H2 SO4 . The optical density of each well was measured at 492 nm in 96-well plate reader and concentration of cytokines in samples was read from a standard curve.

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336 The concentrations of used antibodies where as follows (capture Ab/biotinylated Ab) IL-6, 20, 0.5 µg/mL; IL-12 p40/p70, 20, 0.5 µg/mL; TNF-α, 80, 20 µg/mL. IL-10 was determined by using IL-10 OptEIATM ELISA set as recommended by manufacturer. Sensitivity of Elisa tests IL-6, 15 pg/mL; IL-10, 15 pg/mL; IL-12 p40, 30 pg/mL; TNF-α, 10 pg/mL.

Statistics The two-tailed Student’s t test was used to evaluate the statistical significance of experimental differences between groups with p < 0.05 taken as the minimum level of significance.

Bryniarski, Maresz, Szczepanik, Ptak, and Ptak inhibition, lowest (0.06 µM) still produced 20–30% inhibition of ROI’s synthesis. R-Mf and Oil-Mf were equally sensitive to enzyme treatment. This decreased ability of enzyme-treated Mf to generate ROIs was also observed when the test was postponed for additional 24 h (results not shown) and this was not due to decreased cell viability. It was concluded that since phagocytosis of opsonized zymosan particles by macrophages depends primarily on integrity of CR2, but also FcR or mannose receptors, or all of them, this might indicate that cell surface changes produced by proteolytic enzymes remain not fully repaired even after 48 h. Production of Cytokines by Enzyme-Modified Macrophages

The cell viability measured by trypan blue exclusion test and MTT test immediately after Oil-Mf treatment with high (40 µM), moderate (1.6 µM), and low (0.06 µM) enzyme concentrations are shown in Fig. 1. Treatment with 40 µM of collagenase or trypsin decreased cell viability by 20 or 25%, respectively as compared with control group (100%). At lowest enzyme concentrations (0.06 µM) collagenase did not affect cell viability while trypsin still lowered it by 12%. Simultaneous viability estimations by trypan blue exclusion test showed somewhat higher values ranging in all groups (including control) between 90 and 95% viable cells. Identical results as with oil-Mf were obtained with resident peritoneal macrophages (results not shown). Macrophages which after enzyme treatment we cultured overnight and then used in tests showed in both methods viability ranging between 90 and 100% values of similarly cultured control macrophages

Both collagenase and trypsin treatment affected significantly the production of IL-6 by resident and oilinduced peritoneal Mf. Results from one representative experiment out of three are shown in Fig. 3. Both enzymes induced a significant (7- to 15-fold) increase of IL6 production by R-Mf at high (40 µM) and low (1.6 uM) concentrations, but this effect vanished at lowest concentration (0.06 µM) used. Also enzyme–modified OilMf showed a statistically significant two–threefold increase of IL-6 synthesis with identical dose dependence. The effect of enzyme treatment on synthesis of other cytokines were either limited or absent. Thus collagenase and trypsin only at highest concentrations (40 µM) stimulated slightly the production TNF-α by both populations of macrophages (for R-Mf, no treatment 6 ± 4, collagenase 18 ± 3, trypsin 19 ± 3 pg/mL; for Oil-Mf the corresponding values were 245 ± 5, 374 ± 8, 445 ± 6 pg/mL. At lower enzyme concentrations values returned to control levels). Production of IL-10 and IL-12 remained unaffected over all enzyme concentrations (control values 30 ± 3 pg/mL and 51 ± 4 pg/mL for R-Mf and 61 ± 4 and 173 ± 10 pg/mL for Oil-Mf).

Production of Oxygen Intermediates by Enzyme-Treated Macrophages

Expression of Surface Markers on Collagenase- or Trypsin-Treated Macrophages

Figure 2 shows the results from one representative experiment out of three. Macrophages treated with trypsin or collagenase for 30 min and then cultured overnight show a significantly diminished ability to produce ROIs when stimulated by zymosan. Both enzymes did not differ in their inhibitory properties and inhibition of ROIs synthesis was proportional to the concentration of enzymes used. While the highest concentration (40 µM) caused 70–100%

Non-treated or collagenase- or trypsin-treated Oil-Mf precultured for 24 h were stained with FITC-conjugated anti-F4/80, anti-Mac-3, anti-CD11b mAbs, or with PEconjugated anti-CD32/16, anti-CD23, anti-CD95L, antiCD14, or CD54 mAbs. Results of one such experiment obtained with highest concentration of enzymes (40 µM) are demonstrated in Table 1. At lower (0.06 µM or 1.6 µM) concentrations of enzymes the differences in expression of

RESULTS Viability of Macrophages Treated With Proteolytic Enzymes.

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Modulation of Macrophage Activity by Proteases

Fig. 2. Decreased production of reactive oxygen intermediates (ROIs) by peritoneal macrophages treated with proteolytic enzymes. Resident (R-Mf) and oil-induced macrophages (Oil-Mf) were treated with serial fivefold dilutions of collagenase (coll) or trypsin (tryps) (40, 8, 1.6, 0.3, 0.06 µM) for 30 min. or left untreated. Then to 5 × 105 cells in 0.15 mL nutrient medium lucigenin and zymosan were added and photon emission was measured over 75–100 min (abscissa). For details see Materials and Methods. The results of one out of three experiments are demonstrated. To avoid data overloading only results with high (40 µM), moderate (1.6 µM), and low (0.06 µM) enzyme concentrations are shown. Chemiluminescence (ordinate) is expresed in arbitrary units (RUL). -¥- –nontreated Mf; -•- –nontreated Mf + zymosan; -N- –enzyme treated (40 µM) Mf; -H- –enzyme treated (40 µM) Mf + zymosan; -×- –enzyme treated (1.6 µM) Mf; -∗- –enzyme treated (1.6 µM) Mf + zymosan. -¤- –enzyme treated (0.06 µM) Mf; -°- –enzyme treated (0.06 µM) Mf + zymosan. Vertical rows—Resident, Oil—induced Mf; horizontal rows—collagenase or trypsin treatment.

examined surface markers between treated and nontreated groups of cells were significantly less pronounced. Treatment with collagenase caused fivefold decrease of CD23+ (FcεRII) and over sixfold decrease of CD95L+ (FasL) and Mac-3 Mf and only marginally affected CD11b+ (CR3 positive) macrophages. Moreover, treatment with collagenase caused marginal decrease of CD14+ (LPS receptor) and F4/80 but did not change the percentage of CD54+ (ICAM-1) and CD16+ (Fcγ RII/III) macrophages. Trypsin-treatment produced similar changes. It diminished strongly (five to tenfold) the numbers of CD23+ , CD95L+ , and Mac-3+ positive cells while the influence

on F4/80+ and CD14+ cells was moderate (approximately twofold reduction of positive cells). DISCUSSION Inflammatory foci contain a plethora of different proteases capable of degrading a broad spectrum of substrates including extracellullar matrix proteins and cell membrane-bound proteins (1, 2, 3). These enzymes which belong to distinct functional groups differing in catalytic mechanisms encompass metalloproteases (e.g. collagenase) the main product of Mf, serine proteases, the

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Bryniarski, Maresz, Szczepanik, Ptak, and Ptak

Fig. 3. Production of IL-6 by Mf treated with proteolytic enzymes. Five ×105 nontreated or enzyme treated resident, or oil induced peritoneal macrophages (for details see Legend to Fig. 1 and also Materials and Methods) were cultured in 1 mL of RPMI 1640 medium for 48 h and concentration of interleukin (IL-6) in supernatants was measured by ELISA assay. Table shows the results of one representative experiment out of three and only results with high (40 µM), moderate (1.6 µM), and low (0.06 µM) enzyme concentrations are shown. Each bar represents the mean of three estimations ± SD. Statistical significance in both groups between A and B, C, D, E p < 0.01

hall-mark of neutrophils (e.g. elastase, cathepsin G) and cysteine, and aspartate lysosomal proteases released from damaged cells. In our experiments we have used collagenase and trypsin as representatives of metalloprotease and serine proteases respectively, which although not identical with enzymes present in inflammatory foci show, within groups, common structural domains (10). The results we obtained in vitro with these enzymes give credence to the suggestion that local release of proteases by phagocytic cells can play a regulatory role in the development of inflammatory reactions. Our experiments demonstrate that brief in vitro treatment of Mf with highest concentrations (40 µM) of these proteolytic enzymes decreases immediatelly the cell viability as measured by MTT method up to 20–25% in comparison with nontreated cells while lowest concentrations (0.06 µM) remain without effect. Mf used in tests after 24 h culture show viability ranging between 90 and 100% of control values. Enzyme treatment influences biological activity of Mf in such a way that the production of ROIs is severely impaired while the production of IL-6 is significantly increased. Production of TNF-α was affected only marginally and production of IL-10 and IL-12 remained unchanged. The final outcome was partially dependent on the developmental stage of Mf and on the concentration of enzymes. Thus while nonstimulated resident peritoneal Mf when modified enzymatically produced vigorously high amounts of IL-6, inflammatory oil-Mf were under similar conditions only moderately stimulated. No such relationship was found in ROI’s production and RMf and Oil-Mf were equally sensitive to enzyme handling. These enzyme-dependent mechanisms seem to be highly

effective, since even minute concentrations of enzymes produced significant changes in ROIs synthesis and IL-6 production by Mf and the observed effects were longlasting and could be observed even after 48 h in culture. Our experiments thus may suggest that in in vivo situation Mf newly arriving to the site of inflammation are more easily controlled by environmental influences then already stimulated Mf present in the inflammatory area. It has been shown previously that enzymes, particularly metalloproteases, released by phagocytes cleave many cell membrane molecules (CD2, CD4, CD8, CD23, CD27, CD32, CD43, TNF-α (CD120), and IL-6 (CD126) receptors (2,11,12, 13). Our experiment added to this list Mac-3, CD95L, and CD14 molecules, but presumably many others are similarly (see infra) affected. The change of surface phenotype may significantly influence the cell behavior. For instance such mechanism may be responsible for diminished ROIs synthesis found in our experiments. NADPH oxidase catalysing the generation of ROIs, is composed of several cytosolic and membrane bound proteins which in resting phagocytic cells are segregated. After the cell receives a proper signal (e.g. phagocytosis) cytosolic components translocate to the cell periphery and anchor to membrane-bound proteins, flavocytochrome B complex, to form an active enzyme (4,14). We regard as a possible explanation that the treatment of Mf with proteolytic enzymes causes removal and/or functional impairment of cell surface-bound docking proteins which by impeding the formation of active enzymatic NADPH oxidase complex leads to diminished ROI’s production.

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Modulation of Macrophage Activity by Proteases Table 1. Expression of Cell Surface Markers by Enzyme-Treated Mf Treatment of Mf with

CD32/16-PE

CD23-PE

CD11b-FITC

F4/80-FITC

CD14-PE

CD54-PE

CD95L-PE

Mac3-FITC

No treatment Collagenase Trypsin

93.3 90.3 89.5

59.8 15.0 16.7

92.0 81.5 72.6

87.3 70.3 49.2

91.2 59.7 41.3

92.9 95.1 92.6

45.9 10.0 4.5

76.3 12.0 8.5

Note. Oil-induced macrophages treated with 40 µM of collagenase or trypsin for 30 min were cultured in vitro overnight. Adhering cells were detached with 0.02% EDTA solution, Fcγ R were blocked, and macrophages stained with FITC or PE-conjugated mAbs against cellsurface markers. The percentage of cells considered positive was determined by subtracting from experimental values the percentage of self-fluorescent nonlabelled control cells. The self-fluorescence of nontreated cells vs. cells with blocked Fcγ R was for FITC (525 nm) 5.2 and 3.1% correspondingly and for PE (575 nm) 7.1 and 4.1%. Experimental details are given in Materials and Methods. The Table shows one of two experiments done. Each sample was measured in duplicate. Lower enzyme concentrations produced less pronounced results.

At present the reasons for increased IL-6 release by enzymatically treated macrophages cannot be interpreted unequivocally. One possibility is that it is because of peeling off the membrane-bound cytokines which would make it a purely surface event (13,15–17). This explanation seems rather unlikely since Mf after enzyme-treatment were cultured in vitro overnight before including them into tests. Similarly unlikely is a possibility that high amounts of IL-6 in supernatants of enzyme treated cells were due to release of preformed cytokine from intracellular storage sites since cytokines were not shown to be produced in anticipation. Other possibility we favor is that partial digestion of certain surface receptor molecules alters their spatial configuration and thus may mimic the specific ligand binding. The information is then transmitted into cell interior and translated into expression of relevant gene. Several possible receptor candidates known to be responsible for IL-6 production can be considered (e.g. Fcγ R or CD14). Participation of FcεRII (CD23) is also a possibility since its ligation leads to increased synthesis of several mediators (18,19). Considerning however, that synthesis of other cytokines was either not affected by enzymatic treatment (IL-10, IL-12) or affected only marginally, the mechanism of “enzymatic stress” in the case of IL-6 deserves further study. The observed enzyme-induced shift in macrophage synthetic potential makes biological sense. At certain point in the development of inflammatory reaction it is important to self-limit the tissue-damaging effects of Mf executed by extracellularly exported ROIs and proteolytic enzymes. Our observation suggests that the latter can serve in autocrine or paracrine manner as potent feedback inducers of increased cytokine production and as inhibitors of ROIs synthesis. Inhibition of ROIs production limits their tissue-destructive effects while increased IL-6 synthesis may serve two goals, local and systemic. This cytokine frequently regarded as proinflammatory,

is highly pleiotropic. It downregulates TNF-α synthesis (19,20,21) and stimulates production of acute phase proteins (APP) in the liver, including α1 -antitrypsin, α1 anti-chymotrypsin, and α2 -macroglobulin (5,6,22,23). All these APP are protease inhibitors and thus limit the action of proteolytic enzymes liberated by phagocytic cells. Moreover IL-6 increases release of ACTH by hypophysis, which in consequence stimulates the production of antiinflammatory corticosteroids (22,24). Parenthetically, since only minute concentrations of enzymes are sufficient to affect Mf, the presence of protease inhibitors in inflammatory foci does not necessarily have to neutralize their activity. In summary, our experiments seem to suggest the existence of additional regulatory circuit in which proinflammatory proteases, feedback induce anti-inflammatory mechanisms such as limitation of ROI’s production by macrophages and indirectly increased synthesis of APP. Acknowledgments—This study was supported by a Grant N◦ 4P05A 10417 from Committee of Scientific Research (KBN, Warsaw, Poland) to K. B.

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