Eur. J. Immunol. 1979. 9: 613-619
Abteilung fur Experimentelle Dermatologie, Universitats-Hautklinik, Miinster
Production of fibrinolysis inhibitors by murine macrophages
613
of the activation state in murine macrophages* Macrophages from peritoneal exudates which had been induced by various irritants were cultured, and the supernatants were tested for inhibitors of fibrinolysis using the plasmin-dependent lysis of '2sI-labeled fibrinogen as assay. Washout macrophages and casein or proteose peptone-elicited macrophages were found to release fibrinolysis inhibitors in contrast to lipopolysaccharide or thioglycollate-induced macrophages. The molecular weight of inhibitors was determined at 60000, 45000 and 15000 by Sephadex chromatography. Whereas the inhibitors at 15 000 and 45 000 could be detected in all experiments, the inhibitor at mol. wt. 60000 was not present in all preparations. The isoelectric point of inhibitor I (mol. wt. 15000) and inhibitor I1 (mol. wt. 45000) was determined at 4.15. The proteolytic and esterolytic activity of trypsin and chymotrypsin were both inhibited by each of the two inhibitors. On the other hand, only the proteolytic activity of plasmin could be inhibited. Evidence for the active synthesis of inhibitors by macrophages came from several experiments. Macrophages in serum-free cultures continued to release inhibitors for at least 48 h; normal mouse serum did not contain inhibitors of the same molecular size, and [3H]leucine was incorporated into the inhibitors which were specifically detected by the absorption of plasmin inhibitor complexes to lysyl-Sepharose. Since inhibitors and plasminogen activator could not be detected in the same macrophage culture supernatants, it appears that the production of inhibitors and plasminogen activator by the same macrophage population is mutually exclusive.
1 Introduction
reported by Vassalli and Reich [15]. Apart from its central role of connecting several humoral systems, plasmin is also consid-
Besides their well-known functions as phagocytic cells, the role of macrophages in the immune response as effector and regulatory cells is becoming increasingly recognized [ 1-91. While it has been known for some time that macrophages from different compartments are functionally different [lo], only in recent years the functional heterogeneity of macrophages within the same compartment has been described [lo]. A wide variety of functions could be induced in peritoneal exudate cells by previous injection of various irritants. At present, nothing is known about the mechanism by which various stimuli produce macrophages with different functions. A great number of biochemical functions for elicited or induced macrophages as well as for lymphokine-activated macrophages have been described [ll-201, among them the production of plasminogen activator by thioglycollate-induced peritoneal exudate macrophages, whereas normal washout casein or proteose peptone-elicited macrophages were not found to produce plasminogen activator [20]. Recently, we could show that proteose peptone-elicited macrophages can be induced by lymphokines in vitro to produce plasminogen activator [14], thus establishing a link from cellular immune reactions to humoral systems such as the complement, the fibrinolytic and the kinine-forming system. Similar results have also been
ered to affect the cellular immune response by a direct or indirect mechanism [21]. In the course of our studies on the lymphokine-induced production of plasminogen activator, it was observed that some macrophage culture supernatants not only lacked the plasminogen activator but were even inhibitory to plasmin-dependent fibrinolysis. This observation and the scarcity of reports on protease inhibitor production by macrophages prompted us to investigate this subject more systematically. It was found that normal washout, proteose peptone or casein-elicited macrophages produce fibrinolysis inhibitors in contrast to thioglycollate-induced macrophages which release plasminogen activator.
[I2051] * This study was supported by the Bundesministerium fur Forschung und Technologie (BCT 108). Correspondence: Volker Klimetzek, Abteilung fur Experimentelle Dermatologie, Universitats-Hautklinik, D-4400 Munster, FRG Abbreviations: D-MEM: Dulbecco's modified minimum essential medium FCS: Fetal calf serum LPS: Lipopolysaccharide EBSS: Earl's balanced salt solution PBS: Phosphate-buffered salt solution TPCK: pTosyl-L-phenylalaninechlormethylketon TAME: N-p-tosyl-L-arginine methylester BTEE: N-benzoyl-L-tyrosine ethylester 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979
2 Materials and methods 2.1 Culture media Dulbecco's modified minimum essential medium (D-MEM), supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, penicillin and streptomycin was used throughout. All reagents were purchased from Seromed (Munchen, FRG). Macrophages were cultured either with D-MEM containing 10% fetal calf serum (FCS) or 0.02% lactalbumin hydrolysate (Serva, Heidelberg, FRG). In some experiments, the FCS used was depleted of plasminogen by two cycles of affinity chromatography on L-lysine-Sepharose 4 B and incubated for 2 h at pH 3.2 to destroy serum plasmin inhibitors [22].
2.2 Macrophage cultures BALB/c mice, purchased from the Zentralinstitut fur Versuchstierforschung, Hannover, were kept under conventional 0014-2980/79/0808-0613$02.50/0
614
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C . Sorg
conditions and were used at the age of 1%12 weeks. Macrophages were obtained from the peritoneum of mice injected 3 days earlier with 1 ml of 10% proteose peptone (Difco, Detroit, MI), or 1 ml of 1% casein (Merck, Darmstadt, FRG), or 20 yg lipopolysaccharide (LPS, Difco), or 1 ml of 3% thioglycollate broth (Difco). The cells were seeded in densities ranging from 1.4 x lo5 to 2.3 x lo5 cellsiwell of a microtiter plate (Falcon Plqstics, Oxnard, CA, No. 3040). After 2 h, the nonadherent cells were washed out and the monolayers cultured for another 24 h in D-MEM plus 10% FCS. After 24 h, the macrophage cultures were washed twice with Earle's balanced salt solution (EBSS) and then incubated with 150 pl serum-free D-MEM plus 0.02% lactalbumin hydrolysate for varying times up to 36 h. The supernatants were tested either immediately for inhibitory activity or were stored at - 70°C until use. For preparation of large amounts of supernatants, macrophages were also cultivated in culture flasks (Corning Glass Works, Houghton Pk, Corning, NY, No. 25110) at a density of 8 x lo5 cells/cm3. Serum-free supernatants were obtained as described above.
2.6 Fibrinolysis assay
'251-labeled fibrinogen-coated microtiter plates were prepared as described before [26]. The plates were washed once with PBS and used immediately for the fibrinolysis assay without prior conversion of the fibrinogen to fibrin. One hundred pl of the test solution and 20 p1 of plasminogen or plasmin of the chosen concentration was added. After incubation for 24 h at 37"C, the released radioactivity contained in 100 y1 was counted. Controls were made by incubating the same amount of plasminogen or plasmin in pure medium or buffer. The amount of total digestible 1251-labeledfibrinogen was determined by a 2-h incubation of 10 wells with 0.25% trypsin. The average value was taken as 100%. For simplicity, the term fibrinolysis is used for the lysis of fibrinogen, since the specificity of the enzyme reaction is the same. In experiments studying the specificity of the inhibitors, trypsin (TPCK-treated, 40 U/ mg, Serva) and chymotrypsin (45 Uimg, Serva) were used in the fibrinolysis assay. Twenty yl of trypsin or chymotrypsin in a concentration of 0.1-0.2 pg were incubated in 100 pl or D-MEM, or inhibitor containing supernatants, for 1 h.
2.3 Radiolabeling of macrophage cultures
Proteose peptone-elicited and thioglycollate-induced macrophages were seeded in a density of 8 x lo5 cells/cm2 in culture flasks (Corning, No. 25110). After depletion of nonadherent cells, the macrophages were incubated for 24 h in leucinefree D-MEM containing 200 pCi[3H]leucine (spec. act. 50 Ci/mmol = 1.85 TBq/mmol). After 24 h, the medium was replaced by D-MEM, containing 0.02% lactalbumin hydrolysate and incubated for another 24 h. The supernatants were removed, centrifuged (3500 x g, 15 min) and frozen in aliquots at - 70°C until use.
2.7 Isolation of labeled plasmin inhibitor complexes
Five ml of [3H]leucine-labeled serum-free supernatants were incubated for 15 min at 37 "C with urokinase-activated plasmin (1.25 CU). One ml packed lysyl-Sepharose was then added and incubated for 30 rnin at 4°C with shaking. The reaction mixture was filled into a 2-ml syringe with a cotton plug at the bottom and washed with 0.1 Mphosphate buffer, pH 7.4, until the radioactivity of the effluent was constant. The lysylSepharose was then transferred to a scintillation vial and counted. As control, identical supernatants were processed without plasmin.
2.4 Preparation of cell lysates 2.8 Proteolytic and esterolytic assays
Macrophages cultivated on petri dishes (030 mm, Falcon) were freed of nonadhering material, rinsed by the addition of 10 successive aliquots of 0.9% NaCl which had been warmed to 37°C. One ml of cold phosphate-buffered salt solution (PBS) containing 75 mM sucrose was added to the dishes which were scraped with a rubber policeman. The homogenate was frozen and thawed 5 X . During each thawing, the lysate was forced 20 x through a pasteur pipette. The homogenates were centrifuged (40000 x g, 1 h), and the supernatants were tested for inhibitory activity. 2.5 Plasminogen, plasmin and urokinase
Plasminogen was isolated by affinity chromatography from FCS according to the procedure of Deutsch and Mertz 1231. The plasminogen eluted with 0.2 M eaminocaproic acid was precipitated overnight with 3.1 g ammonium sulfate/lO ml. After centrifugation (3500 x g, 10 rnin), the precipitate was dissolved in 0.05 M Tris-0.02 M lysine buffer, pH 9.0, [24] at a concentration of 0.2-0.4 CU/ml[25]. Aliquots were stored frozen at -70°C. Plasmin was obtained by activation with urokinase. Urokinase (Serono, Freiburg, FRG) was dissolved in 0.9% NaCl at a concentration of 250 CTA Uiml and stored frozen; 0.1 U was needed to activate 20 p1 plasminogen. This concentration of urokinase contained no fibrinolytic activity.
The caseinolytic assay of plasmin was performed with 14C-labeled N,N-dimethylcasein, prepared as described by Drucker [27]. The hydrolysis of N-p-tosyl-L-arginine methylester (TAME) by trypsin and of N-benzoyl-L-tyrosine ethylester (BTEE) by chymotrypsin were measured by a modification of the esterolytic methods of Hummel [28]. The hydrolysis of H-D-valyl-leucyl-lysyl-p-nitroanilid(S: 2251, which was donated by Kabi, Stockholm, Sweden) by plasmin was measured as follows: to 600 pl D-MEM or inhibitor-containing supernatants Tris-HC1 was added to a concentration of 0.05 M, pH 7.4. The mixture was incubated with 100 pl plasmin (0.25 CU/ml; Kabi) for 5 rnin at 37°C to which 200 y10.875 mM of the substrate was added. The extinction of released paranitroaniline was measured at 405 nm. 2.9 Sephadex chromatography
One hundred to 150 ml of macrophage culture supernatant was centrifuged (25 000 x g, 20 min) and concentrated to 2 mi over an Amicon UM-membrane (exclusion limit 1000 mol. wt.). After a second centrifugation (100000 x g, 30 min), the concentrate was fractionated on Sephadex G-150 columns (1.5 X 120 cm); 0.1 M phosphate buffer containing 0.3 M NaCl was used as eluant. Fractions of 1 ml were collected; 100 pi of
Eur. J. Immunol. 1979.9: 613-619
each fraction was tested for inhibitory activity. The active fractions were pooled and stored frozen at -70°C. 2.10 Isoelectric focusing Isoelectric focusing of the inhibitors was carried out in a sucrose gradient with a micro-preparative apparatus, as described by Sorg and Bloom [29]. At the end of the separation, 100 pl fractions were collected, diluted with 100 pl distilled water and the pH determined in every second sample. To remove the Ampholines (Pharmacia, Uppsala, Sweden), the fractions were dialyzed extensively for 48 h at 4°C against 0.1 M phosphate buffer, pH 7.4, which was frequently changed. The low mol. wt. inhibitor was dialyzed over an Amicon UM 2 membrane.
3 Results
Production of fibrinolysis inhibitors by murine macrophages
615
Table 1. Inhibitory activities of serum-free supernatants from proteose peptone-elicited exudates of single BALB/c mice")
Mouse
No.
Dilutions of macrophage culture supernatants 1:l 1 :2 1:4
48 46 77 34
81 62 43
Inhibition of fibrinolysis (96) 31 36 62 16 13 43 31
24 24 51 5 52 30 1.1
One hundred +I of supernatants were tested with urokinase (2.5 X CU)-activated plasminogen. The fibrinolytic activity of the urokinase-formed plasmin was set at 100%. Each value is the mean of three determinations. Maximal deviation was within f 8%.
3.1 Assay of fibrinolysis inhibitors
v. I The presence of either fibrinolysis inhibitors or plasminogen activators in a culture supernatant can be assayed in the same test, if a mixture of plasmin and plasminogen is used, which by itself causes a lysis of 12sI-labeledfibrinogen up to 50%. When inhibitors are present, the fibrinolytic activity of plasmin will be reduced, whereas plasminogen activator will activate plasminogen and increase the fibrinolysis up to 100%. Plasminogen isolated from FCS by affinity chromatography on lysylSepharose, according to the procedure of Deutsch and Mertz [23], always contains a certain amount of active plasmin and, therefore, can be used for the simultaneous detection of plasminogen activator and fibrinolysis inhibitors. Since it was found (data not shown) that the use of L251-labeledfibrinogen increased the sensitivity of the assay about 4-fold, '251-labeled fibrinogen-coated plates were used throughout without prior 00 conversion to fibrin. In order to detect either plasminogen 25 12.5 6.25 activator or fibrinolysis inhibitors in macrophage culture 1 x 10-3 cu plasminogen supernatants, the following concentrations of plasminogen were used: for the detection of plasminogen activator, a Figure 1. Effects of various macrophage culture supernatants concentration of plasminogen/plasmin was chosen (6 x lo5 cells/cm*) on the fibrinolysis of three different concentrations (2.5 x 10-3CU) [25],which showed no more fibrinolytic activ- of plasmin/plasminogen (C---a). Each point is the mean value of 3 ity, but degraded nearly 100% of fibrinogen after complete determinations. The background fibrinolytic activity was 6%. Peritoconversion of plasminogen to plasmin with either plasminogen neal macrophages: Thio = thioglycollate-induced; LPS = lipopolysacactivator or urokinase. For the detection of fibrinolysis charide-induced; P.P. = proteose peptone-elicited; Cas. = caseininhibitors, the same concentration of plasminogeniplasmin was elicited; W. out = Normal washout. used after conversion of plasminogen to plasmin with urokinase. For the simultaneous detection of plasminogen activators and fibrinolysis inhibitors, a concentration of plasminogeni activator, as described by others [20]. The amount of secreted plasmin was chosen (15 x lop3 CU) which degraded about inhibitory activity of casein or proteose peptone-elicited 50% of the fibrinogen. These concentrations of plasminogeni peritoneal macrophages varied from one experiment to plasmin had to be determined for each batch since the ratio of another. This variation seemed to depend on the physical state of the mice, since the amount of secreted inhibitory activity plasminogen to plasmin was variable. was also different in supernatants of macrophages from individual mice within the same experiment (Table 1). 3.2 Secretion of fibrinolysis inhibitors by peritoneal exudate macrophages 3.3 Chemical characterization of inhibitory activities Inhibitory activity was detected in normal washout and in proteose peptone or casein-elicited macrophages (Fig. 1). LPS- 3.3.1 Sephadex chromatography induced macrophages were not found to secrete inhibitory activity or plasminogen activator. On the other hand, thiogly- Serum-free supernatants of casein and proteose peptone-elicollate-induced macrophages readily produced plasminogen cited macrophages were fractionated on Sephadex G-150.
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C. Sorg
616
Table 2. Recovery of inhibitory activity from a serum-free supernatant of proteose peptone-elicited macrophages after separation on Sephadex G-150
Quantity (ml) 111
Supernatant Inhibitor I Inhibitor I1 Inhibitor 111
125 24 10
Inhibitory activity (%) KCCOVCI!. Dilutions"' ('+) 112 I : J 1 . 8 I : I ~
72'" 77 73 6s
x
56
17
-
-
I00
7x 71 51
6s
5-1 2x
32
44
-
I6
-
-
6
48 20
a) Dilutions were done with elution buffer. b) Mean of three determinations. The fibrinolytic activity of urokinase-activated plasmin was the same as in Fig. 2; mean deviation: k 9%.
Table 3. Inhibition of proteolytic and esterolytic activity of plasmin, trypsin and chymotrypsin by unfractionated supernatants and isolated inhibitor I and I1 a) Sp = unfractionated supernatant. Substrates Plasmin Trypsin bl I = Inhibitor I. sl, II c) I1 = Inhibitor 11. spa' l t ' l I[') sp I I1 d) ++ = completely inhibited. '2'I-labelctl hhrinogcn ++"' + + + + ++ ++ ++ ++ ++ ++ e) n.d. = not done. 'I'AME n.d.'l +'I + t 11.d. f) + = partial inhibition. H'I'EE n.d. n.d. + + g) S : 2251 = H-D-valyl-leucyl-lysyl-I I I s : 225 I P' n.d. n.d. p-nitroanilid. h) - = no inhibiton.
,
Each fraction was assayed for inhibition of the fibrinolytic activity of plasmin. Three activities at the mol. wt. ranges of 60 000, 45 000 and 15 000 were detected (Fig. 2). The recovery of the inhibitory activity applied to the column was about 70% (Table 2). The activity at mol. wt. 60000 was not found in every experiment and was therefore not accessible for extensive characterization. The activity at mol. wt. range 15 000 was hence designated as inhibitor I and at 45000, as inhibitor 11. Supernatants of LPS and thioglycollate-induced macrophages, fractionated and assayed in an identical way, did not contain detectable amounts of inhibitory activity in any fraction (data not shown).
3.3.2 Isoelectric focusing Pooled fractions of the Sephadex G-150 chromatography at the mol. wt. range 15000 (inhibitor I) and 45 000 (inhibitor 11)
were subjected to isoelectric focusing in a sucrose gradient. As shown in Fig. 3 for inhibitor 11, the inhibitory activity peaked at pH 4.1 to 4.3. Isoelectric focusing of inhibitor I (data not shown) gave identical results.
3.3.3 Stability and specificity of inhibitors The activity of both inhibitors was stable during incubation for 1 h at 56 "C and was completely destroyed after incubation for 1 h at 70°C. This, together with the labeling data (Sect. 3.4.) provides evidence that the inhibitors are in major parts of protein nature. In order to determine the sepcificity of inhibitors, supernatants of proteose peptone or casein-elicited macrophages were tested not only in the plasmin but also in the trypsin and chymotrypsin-dependent lysis of fibrinogen (Fig. 4). One hundred p1 of the supernatant obtained from proteose peptone-elicited macrophages, cultivated at a density of 8 X lo5 cells/cm2, caused a 50% inhibition of 0.25 cig chymotrypsin and of 0.3 pg trypsin. When the isolated inhibitors I and I1 were assayed, the proteolytic activity of chymotrypsin and trypsin was similarly inhibited (Table 3). Inhibitor I and % 70
F
In .-
(BSA
il
70
80
(OA
90 100 110 fraction number
-x 50VI
(Chi 120
130
140
150
Figure 2. Sephadex G-150 chromatography of culture supernatants of proteose peptone-elicited peritoneal macrophages. (BSA: bovine serum albumin, mol. wt. 67000; OA: ovalbumin, mol. wt. 45000; Chy: chymotrypsinogen A 25000; Cyt.: Cytochrome C, mol. wt. 12500). Each point is the mean value of three determinations. The fibrinolytic activity of the used urokinase-activated plasmin (2.5 X CU) was 93 rf: 8%.
.-L
e
L
2 4
30
fraction number
Figure 3. Isoelectric focusing (pH 3.5-10) of inhibitor I1 (mol. wt. 45 000) after Sephadex chromatography. The fibrinolytic activity of CU) is shown by the the used urokinase-activated plasmin (2.5 X broken line. pH gradient @---A).
Eur. J. Immunol. 1979. 9: 613-619
Production of fibrinolysis inhibitors by murine macrophages
617
rophages cultured for the first 24 h in the presence of normal FCS contained the highest inhibitory activity. In the other type of supernatants, the inhibitory activity was decreased by about 30-50%. Similar results were obtained when the cell lysates of the corresponding cultures were tested. From these data, it is not clear whether the inhibitory activity represents absorbed and released, or synthesized material. The decrease in inhibitor release by cultures kept either serum-free or with acid-treated FCS might be due to the deteriorating culture conditions or to a gradual change in the plasminogen activatorproducing state induced by the culture conditions. kg trypsin
wg chymotrypsin
Figure 4. Effect of culture supernatants (H from )proteose peptone-elicited macrophages (8 X 10' cells/cm2) on the fibrinolytic activity of trypsin (a) and chymotrypsin (b). Activity of the enzymes alone was measured in D-MEM (H).
11, as well as the unfractionated supernatants, inhibited the esterolysis of TAME by trypsin and of BTEE by chymotrypsin. In this case, however, no complete inhibition could be obtained, neither by the supernatants nor by the isolated inhibitors. In both cases, only 50% of the esterolytic activity was inhibited. If the inhibitors were incubated prior to the experiment for 1 h at 70°C, no inhibition of the esterolytic activity of trypsin and chymotrypsin was observed, which is in line with the experiments on the stability of inhibitors described above. 3.4 Synthesis of inhibitors by macrophages
After the detection and chemical characterization of inhibitory activities in supernatants of certain macrophage cultures, the question was still open whether the inhibitory material was actively synthesized or just released material which had been previously absorbed from the serum in vivo or from the serum in the culture medium. In order to clarify this question, the following experiments were performed. Macrophages were cultivated for the first 24 h in D-MEM containing either 10% FCS, 10% acid-treated, inhibitory-free FCS or 0.2% albumin. All three types of cultures were then transferred to serum-free medium and cultured for another 24 h. When the supernatants were tested for inhibitory activity (Table 4), it was always found that supernatants of mac-
If, however, the inhibitory activity had been previously absorbed from serum either in vivo or from the FCS in vitro, the same inhibitory activities should be detected in normal mouse serum or in FCS, as well as in macrophage culture supernatants after fractionation. Normal mouse serum and FCS were therefore fractionated and tested for inhibitory activity. When the fractions of FCS were tested for inhibition of plasmin, two major peaks of inhibitory activity were found with mol. wts. higher than 80000 (data not shown). Occasionally, a small amount of an additional inhibitory activity with a mol. wt. of 15000-20000 was found which was not dialyzable, in contrast to the inhibitor I from macrophage culture supernatants, and which was lost after a dilution step of 1: 2. No inhibitory activity was ever found at rnol. wts. 25 000-80000. When mouse serum was analyzed the same way, high mol. wt. inhibitors were readily detected but not low mol. wt. inhibitors in the range of 15 000-60 000. These experiments, in connection with the data shown in Table 4, are evidence that the inhibitors are not absorbed from the serum in vivo or from the FCS used in culture medium. Since the experiments described above provided only circumstantial evidence, we investigated whether the inhibitory material could be internally labeled by a radioactive amino acid. Thioglycollate-induced and proteose peptone-elicited macrophages, known to produce either plasminogen activator or inhibitory activity, were therefore labeled with ['HH]leucine. The radiolabeled inhibitory material was detected by specific absorption of the plasmin-inhibitor complex to lysyl-Sepharose. As shown in Table 5 , thioglycollate-induced macrophages do not produce labeled material that binds specifically to plasmin, whereas proteose peptone-elicited macrophages produce material which is specifically bound, amounting to about 6% of the total labeled material which was not found when plasmin was omitted. Experiments performed
Table 4. Inhibitory activities of serum-free supernatants and cell lysates of macrophages after preceding culture in medium containing either normal FCS (n-FCS), inhibitor-free FCS (at-FCS) or no serum
Pcritoncal rnacrophagcs elicited by
Serum uscd in culture
Su pcrna t ant I:l
1::
I:4
C'cll I!\alc 1:l 1.2
Inhihition of (ihrinolvsi\ ( ' i Proteosc peptone
ciwin .
(J.
'
n-FCS at-FCS No," seruni
73
hX
23
sx
56 3x
37 II
Ih
41 33
n-FCS ;lt-FC'S No"' wrum
77 35 73
53
17
15 0
0
II
0
11.d.
(1
)
20 I2 18 I1.ll ."
.d
'
a) The supernatant was tested after 24 h of culture. b) not done.
618
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C. Sorg
Table 5. Detection of radiolabeled inhibitors by specific binding of plasmin inhibitor complexes to lysyl-Sepharose PcritDneal
Plasmin
macrophages induocdlelicited
supern3tlysylant Srpharose (rpm x fcprn) 10 ’)
by
Tluoglycollatc
’I‘otal Radioac- ci: of rulal radioiictiv- tiviry radioscit); of hound to ti\i;ity
-
1.13
t
1.21
29,168 S1.YKS
0.42
c
I .32 1.33
40.272 81!.Wo‘
0.30 6 1
Proteose peplone
(0.25
to recover the inhibitory activity have as yet been unsuccessful because the plasmin-inhibitor complex could not be dissociated without loss of biochemical activity.
4 Discussion Using the plasmin-dependent lysis of ‘2sI-labeled fibrinogen, it could be shown that nonstimulated washout as well as proteose peptone or casein-elicited macrophages release inhibitors of fibrinolysis, in contrast to thioglycollate-induced peritoneal macrophages which produce plasminogen activator but no inhibitors. The inhibitor production was also not changed after phagocytosis of latex beads (unpublished observations). Macrophages from LPS-induced peritoneal exudate cells have been described to release plasminogen activator only after phagocytosis of latex beads [30]. Inhibitors were also missed in culture supernatants of LPS-induced macrophages before and after phagocytosis of latex beads. Inhibitors and plasminogen activator have so far not been observed to be produced simultaneously by cells of the same culture. The possibility that both activities are produced and that the differences are quantitaive in nature could be ruled out by experiments where the supernatants were fractionated on Sephadex. Plasminogen activator and inhibitors which have different mol. wts. were not detected in the same culture supernatants. By Sephadex chromatography, the mol. wt. of inhibitors was determined at 60000, 45 000 and 15 000, which clearly shows that the inhibitors are different from az-macroglobulin which has been described to be produced by human rnonocytes [311. The inhibitors were found to block the proteolytic and esterolytic activity of trypsin and chymotrypsin. On the other hand, only the proteolytic activity of plasmin was inhibited. Whether the inhibitors can also inhibit plasminogen activator is not known at present, since the determination of the split product of plasminogen requires different methods [32]. It certainly would be most interesting to see whether inhibitor-producing macrophages are the direct antagonists of the plasminogen activator-producing rnacrophages. An important question was whether the inhibitors are actively synthesized or whether they are material which had been taken UP by macrophages from the Serum in viuO 01‘from the serum added to the culture medium. Evidence for an active synthesis came from the chemical characteristics of the
inhibitors which are different compared to all known serum protease inhibitors (331. Along the same lines, it was shown that mouse serum did not contain any detectable amounts of low-mol. wt. inhibitors. Peritoneal macrophages which were cultured in serum-free medium nevertheless released inhibitors of lower molecular size. On the other hand, the possibility exists that macrophages take up high-mol. wt. inhibitors which are subsequently released in degraded though active form. Strong evidence for a synthesis of inhibitors came from experiments using radioactive leucine for internal labeling of inhibitors. The plasmin-inhibitor complexes were detected by specific absorption to lysyl-Sepharose. The amount of inhibitors produced is quite substantial with 6% of the total macromolecular counts in the culture supernatants. Since the absorbed labeled material could not be desorbed for further functional and chemical characterizations, the final proof for identity of labeled material and of inhibitory activity could not be achieved. The possibility that the labeled material is just a substrate for plasmin seems unlikely because of the negative control experiment performed with supernatants from thioglycollate-induced macrophages. From these data, it appears that the release of fibrinolysis inhibitors is a property of macrophages in a certain functional state. Others have reported a wide variety of functions which are enhanced or newly acquired by macrophages after exposure to lymphokines [7,11-15,261. An interesting question, therefore, would be whether the production of fibrinolysis inhibitors is modulated under the influence of lymphokines. In recent experiments (V. Klimetzek and C. Sorg, in preparation) it was found that inhibitor production by macrophages is reduced or even completely abolished after exposure to lymphokines from mitogen-stimulated spleen cells. Cultures of casein or proteose peptone-elicited macrophages could even be converted from the inhibitor to the plasminogen activatorproducing state. Whether the release of plasminogen activator is a function of “activated” macrophages or a constitutive property of macrophages in a certain maturation state, is currently being investigated. Studying the differentiation of bone marrow cells into macrophages in a liquid culture system, evidence has been obtained suggesting that plasminogen activator-producing macrophages can switch to the inhibitorproducing state in the course of differentiation [34]. W e thank Mr. Erhard Kreinjobst and Ms.Brigitte Brokmeier for expert technical help. Received February 24, 197s; in revised form March 23, 1979.
5 References 1 Van Furth, R. (Ed.), Mononuclear Phagocytes in Immunity, Infection and Pathology, Blackwell Scientific Publications. Oxford 1975. 2 Mackaness, G. B., in Mudd, S. (Ed.), Infectious Antigens and Host Reactions, Saunders, Philadelphia 1970, p. 61. 3 Krahenbuhl, J . and Remington, I. S . , Infect. Immun. 1972.4: 337. 4 Rosenthal, A. S . , Blake, J. T., Ellner, J. J . , Greineder, D. K. and Lipsky, P. E., in Nelson, D . S. (Ed.), Immunobiology of the Macrophage, Academic Press, New York 1976, p. 131. 5 Rosenstreich, D. L. and Oppenheim, J. J., in Nelson, D. S. (Ed.), Immunobiology of the Macrophage, Academic Press, New York 1976, p, 162, 6 Gery, J., Gershon, R. K. and Waksman, B. H., J . Exp. Med. 1972. 136: 128. 7 Neumann, C. and Sorg, C., Eur. J . Immunol. 1977. 7: 719.
Eur. J. Immunol. 1979.9: 619-625
Lipopolysaccharide and lipid A-induced human B cell activation
8 Nelson, D. S . , in Nelson, D. S. (Ed.), Immunobiology ofthe Macrophage, Academic Press, New York 1976, p. 235. 9 David, J. R., Fed. Proc. 1975. 34: 1730. 10 Walker, W. S., in Nelson, D. S . (Ed.), lmmunobiology of the Macrophage, Academic Press, New York 1976, p. 91. 11 Remold-O'Donnell, E . and Remold, H. G., J . Biol. Chem. 1974. 249: 3622. 12 Hammond, N. E. and Dvorak, H. S., J . Exp. Med. 1972. 136: 1518. 13 Wahl, L. M., Wahl, S. M., Mergenhagen, S. E. and Martin, G. R., Science 1975. 187: 261. 14 Klimetzek, V. and Sorg, C., Cell. Immunol. 1976. 27: 350. 15 Vassalli, J. D. and Reich, E., J . Exp. Med. 1977. 145: 429. 16 Karnovsky, M. L., Lazdins, J., Drath, D. and Harper, A,, Ann. N Y Acad. Sci. 1975. 256: 266. 17 Allison, A. C. and Davies, P., in van Furth, R. (Ed.), Mononuclear Phagocytes in Immunity, Infection and Pathology, Blackwell Scientific Publications, Oxford 1975, p. 487. 18 Pantalone, R. N. and Page, R. C., Proc. Nut. Acad. Sci. USA 1975. 72: 2091. 19 Nathan, C. F., Karnovsky, M. L. and David, J. R., J . Exp. Med. 1971. 133: 1356. 20 Unkeless, J . C., Gordon, S. and Reich, E., J . Exp. Med. 1974. 139: 834.
C. I. Edvard Smitho, Lennart Hammarstrom', A. Graham BirdoA, Takao Kunori ,Bjorn Gustafsson' and Tord Holme'
Department of Immunobiology, Wallenberglaboratory', Department of Bacteriology', Karolinska Institute, Stockholm and Transplantation Immunology Laboratory, Huddinge Hospital, Huddinge'
21 Newcomb, E. W., Silverstein, S. C. and Silagi, S . , J . Cell. Physiol. 1978.95: 169. 22 Unkeless, J. C . , Tobia, A . , Ossowski, L., Quigley, J. P., Rifkin, D. B. and Reich, E., J . Exp. Med. 1973. 137: 85. 23 Deutsch, D. G . and Mertz, E . T., Science 1970. 170: 1095. 24 Summaria, L., Arzadon, L., Bernabe, P. and Robbins, K. C., J , Biol. Chem. 1972. 247: 4691. 25 Remmert, L. F. and Cohen, P. P., J . Biol. Chem. 1949. 181: 431. 26 Klimetzek, V. and Sorg, C., Eur. J . Immunol. 1977. 7: 185. 27 Drucker, H., Anal. Biochem. 1972. 46: 598. 28 Hummel, B. C. W., Can. J . Biochem. Physiol. 1959. 37: 1393. 29 Sorg, C. and Bloom, B. R . , J . Exp. Med. 1973. 137: 148. 30 Gordon, S., Unkeless, J. C. and Cohn, Z. A., J . Exp. Med. 1974. 140: 995. 31 Hovi, T., Mosker, D. and Vaheri, A , , J . Exp. Med. 1977. 145: 1580. 32 Dano, K. and Reich, E . , in Reich E., Rifkin, D. B. and Shaw, E. (Eds.), Proteases and Biological Control, Cold Spring Harbor Conferences on Cell Proliferation 1975, vol. 2, p. 357. 33 Wiman, B. and Collen, D., Nature 1978. 272: 549. 34 Sorg, C., Neumann, C., Klimetzek, V. and Hannich, D., in van Furth, R . (Ed.), Mononuclear Phagocytes - Functionaf Aspects, Martinus Nijhoff Medical Division, The Hague, Boston, London 1979, in press.
Lipopolysaccharide and lipid A-induced human blood lymphocyte activation as detected by a protein A plaque assay* Various purified cell wall lipopolysaccharides (LPS) from gram-negative bacteria and derivatives of these LPS were tested for their stimulatory capacity for human peripheral blood cells. Immunoglobulin (Ig) production was tested by an indirect plaque-forming cell assay using Staphylococcus aureus protein A-coupled erythrocytes and specific anti-Ig as developing serum. This method allows the detection of the majority of cells secreting Ig of a single class, and the number of plaque-forming cells detected are approximately 10CL1000 times the amount obtained using normal sheep red cells as targets. LPS containing the 0 antigen-specific chain, as well as mutant products only containing lipid A and ketodeoxyoctonate trisaccharide, could induce cell division and antibody synthesis. The polypeptide antibiotic polymyxin B was found to inhibit LPS-induced activation. Furthermore, purified lipid A, comulexed with bovine serum albumin, was also found to activate human peripheral blood B cells. These findings demonstrate that human peripheral blood lymphocytes can be activated by LPS and also indicate that lipid A is the active part of these molecules. [I 24101
* This work was supported by the Swedish Cancer Society and the A
619
Torsten and Ragnar Soderberg Foundation. Radcliffe Travelling Fellow, Oxford University, GB.
Correspondence: C. I. Edvard Smith, Department of Immunobiology, Wallenberglaboratory, Lilla Frescati, S-104 05 Stockholm 50, Sweden Abbreviations: BSA: Bovine serum albumin BSS: Balanced salt solution LPS: Lipopolysaccharide PFC: Plaque-forming cell(s) PPD: Purified protein derivative of tuberculin PWM: Pokeweed mitogen [jH]dThd: Tritiated thymidine PBL: Peripheral blood lymphocytes SRBC: Sheep red blood cells 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979
1 Introduction Lipopolysaccharides (LPS) have been extensively used in experimental immunology due to their lymphocyte-activating properties [l]. These mitogens have been shown to activate resting mouse B lymphocytes to proliferation, maturation and immunoglobulin (Ig) secretion in a polyclonal fashion [ 2 ] and have thus been designated polyclonal B cell activators. Approximately one-third of all mouse splenocytes are reactive to LPS [3], and the ability to respond is dependent on a gene allocated on chromosome 4 [4]. In the large majority of reports dealing with LPS-induced activation, the effect on mouse splenocytes has been studied. 00l4-2980/79/0808-0619$02.50/0
Abteilung fur Experimentelle Dermatologie, Universitats-Hautklinik, Miinster
Production of fibrinolysis inhibitors by murine macrophages
613
of the activation state in murine macrophages* Macrophages from peritoneal exudates which had been induced by various irritants were cultured, and the supernatants were tested for inhibitors of fibrinolysis using the plasmin-dependent lysis of '2sI-labeled fibrinogen as assay. Washout macrophages and casein or proteose peptone-elicited macrophages were found to release fibrinolysis inhibitors in contrast to lipopolysaccharide or thioglycollate-induced macrophages. The molecular weight of inhibitors was determined at 60000, 45000 and 15000 by Sephadex chromatography. Whereas the inhibitors at 15 000 and 45 000 could be detected in all experiments, the inhibitor at mol. wt. 60000 was not present in all preparations. The isoelectric point of inhibitor I (mol. wt. 15000) and inhibitor I1 (mol. wt. 45000) was determined at 4.15. The proteolytic and esterolytic activity of trypsin and chymotrypsin were both inhibited by each of the two inhibitors. On the other hand, only the proteolytic activity of plasmin could be inhibited. Evidence for the active synthesis of inhibitors by macrophages came from several experiments. Macrophages in serum-free cultures continued to release inhibitors for at least 48 h; normal mouse serum did not contain inhibitors of the same molecular size, and [3H]leucine was incorporated into the inhibitors which were specifically detected by the absorption of plasmin inhibitor complexes to lysyl-Sepharose. Since inhibitors and plasminogen activator could not be detected in the same macrophage culture supernatants, it appears that the production of inhibitors and plasminogen activator by the same macrophage population is mutually exclusive.
1 Introduction
reported by Vassalli and Reich [15]. Apart from its central role of connecting several humoral systems, plasmin is also consid-
Besides their well-known functions as phagocytic cells, the role of macrophages in the immune response as effector and regulatory cells is becoming increasingly recognized [ 1-91. While it has been known for some time that macrophages from different compartments are functionally different [lo], only in recent years the functional heterogeneity of macrophages within the same compartment has been described [lo]. A wide variety of functions could be induced in peritoneal exudate cells by previous injection of various irritants. At present, nothing is known about the mechanism by which various stimuli produce macrophages with different functions. A great number of biochemical functions for elicited or induced macrophages as well as for lymphokine-activated macrophages have been described [ll-201, among them the production of plasminogen activator by thioglycollate-induced peritoneal exudate macrophages, whereas normal washout casein or proteose peptone-elicited macrophages were not found to produce plasminogen activator [20]. Recently, we could show that proteose peptone-elicited macrophages can be induced by lymphokines in vitro to produce plasminogen activator [14], thus establishing a link from cellular immune reactions to humoral systems such as the complement, the fibrinolytic and the kinine-forming system. Similar results have also been
ered to affect the cellular immune response by a direct or indirect mechanism [21]. In the course of our studies on the lymphokine-induced production of plasminogen activator, it was observed that some macrophage culture supernatants not only lacked the plasminogen activator but were even inhibitory to plasmin-dependent fibrinolysis. This observation and the scarcity of reports on protease inhibitor production by macrophages prompted us to investigate this subject more systematically. It was found that normal washout, proteose peptone or casein-elicited macrophages produce fibrinolysis inhibitors in contrast to thioglycollate-induced macrophages which release plasminogen activator.
[I2051] * This study was supported by the Bundesministerium fur Forschung und Technologie (BCT 108). Correspondence: Volker Klimetzek, Abteilung fur Experimentelle Dermatologie, Universitats-Hautklinik, D-4400 Munster, FRG Abbreviations: D-MEM: Dulbecco's modified minimum essential medium FCS: Fetal calf serum LPS: Lipopolysaccharide EBSS: Earl's balanced salt solution PBS: Phosphate-buffered salt solution TPCK: pTosyl-L-phenylalaninechlormethylketon TAME: N-p-tosyl-L-arginine methylester BTEE: N-benzoyl-L-tyrosine ethylester 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979
2 Materials and methods 2.1 Culture media Dulbecco's modified minimum essential medium (D-MEM), supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, penicillin and streptomycin was used throughout. All reagents were purchased from Seromed (Munchen, FRG). Macrophages were cultured either with D-MEM containing 10% fetal calf serum (FCS) or 0.02% lactalbumin hydrolysate (Serva, Heidelberg, FRG). In some experiments, the FCS used was depleted of plasminogen by two cycles of affinity chromatography on L-lysine-Sepharose 4 B and incubated for 2 h at pH 3.2 to destroy serum plasmin inhibitors [22].
2.2 Macrophage cultures BALB/c mice, purchased from the Zentralinstitut fur Versuchstierforschung, Hannover, were kept under conventional 0014-2980/79/0808-0613$02.50/0
614
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C . Sorg
conditions and were used at the age of 1%12 weeks. Macrophages were obtained from the peritoneum of mice injected 3 days earlier with 1 ml of 10% proteose peptone (Difco, Detroit, MI), or 1 ml of 1% casein (Merck, Darmstadt, FRG), or 20 yg lipopolysaccharide (LPS, Difco), or 1 ml of 3% thioglycollate broth (Difco). The cells were seeded in densities ranging from 1.4 x lo5 to 2.3 x lo5 cellsiwell of a microtiter plate (Falcon Plqstics, Oxnard, CA, No. 3040). After 2 h, the nonadherent cells were washed out and the monolayers cultured for another 24 h in D-MEM plus 10% FCS. After 24 h, the macrophage cultures were washed twice with Earle's balanced salt solution (EBSS) and then incubated with 150 pl serum-free D-MEM plus 0.02% lactalbumin hydrolysate for varying times up to 36 h. The supernatants were tested either immediately for inhibitory activity or were stored at - 70°C until use. For preparation of large amounts of supernatants, macrophages were also cultivated in culture flasks (Corning Glass Works, Houghton Pk, Corning, NY, No. 25110) at a density of 8 x lo5 cells/cm3. Serum-free supernatants were obtained as described above.
2.6 Fibrinolysis assay
'251-labeled fibrinogen-coated microtiter plates were prepared as described before [26]. The plates were washed once with PBS and used immediately for the fibrinolysis assay without prior conversion of the fibrinogen to fibrin. One hundred pl of the test solution and 20 p1 of plasminogen or plasmin of the chosen concentration was added. After incubation for 24 h at 37"C, the released radioactivity contained in 100 y1 was counted. Controls were made by incubating the same amount of plasminogen or plasmin in pure medium or buffer. The amount of total digestible 1251-labeledfibrinogen was determined by a 2-h incubation of 10 wells with 0.25% trypsin. The average value was taken as 100%. For simplicity, the term fibrinolysis is used for the lysis of fibrinogen, since the specificity of the enzyme reaction is the same. In experiments studying the specificity of the inhibitors, trypsin (TPCK-treated, 40 U/ mg, Serva) and chymotrypsin (45 Uimg, Serva) were used in the fibrinolysis assay. Twenty yl of trypsin or chymotrypsin in a concentration of 0.1-0.2 pg were incubated in 100 pl or D-MEM, or inhibitor containing supernatants, for 1 h.
2.3 Radiolabeling of macrophage cultures
Proteose peptone-elicited and thioglycollate-induced macrophages were seeded in a density of 8 x lo5 cells/cm2 in culture flasks (Corning, No. 25110). After depletion of nonadherent cells, the macrophages were incubated for 24 h in leucinefree D-MEM containing 200 pCi[3H]leucine (spec. act. 50 Ci/mmol = 1.85 TBq/mmol). After 24 h, the medium was replaced by D-MEM, containing 0.02% lactalbumin hydrolysate and incubated for another 24 h. The supernatants were removed, centrifuged (3500 x g, 15 min) and frozen in aliquots at - 70°C until use.
2.7 Isolation of labeled plasmin inhibitor complexes
Five ml of [3H]leucine-labeled serum-free supernatants were incubated for 15 min at 37 "C with urokinase-activated plasmin (1.25 CU). One ml packed lysyl-Sepharose was then added and incubated for 30 rnin at 4°C with shaking. The reaction mixture was filled into a 2-ml syringe with a cotton plug at the bottom and washed with 0.1 Mphosphate buffer, pH 7.4, until the radioactivity of the effluent was constant. The lysylSepharose was then transferred to a scintillation vial and counted. As control, identical supernatants were processed without plasmin.
2.4 Preparation of cell lysates 2.8 Proteolytic and esterolytic assays
Macrophages cultivated on petri dishes (030 mm, Falcon) were freed of nonadhering material, rinsed by the addition of 10 successive aliquots of 0.9% NaCl which had been warmed to 37°C. One ml of cold phosphate-buffered salt solution (PBS) containing 75 mM sucrose was added to the dishes which were scraped with a rubber policeman. The homogenate was frozen and thawed 5 X . During each thawing, the lysate was forced 20 x through a pasteur pipette. The homogenates were centrifuged (40000 x g, 1 h), and the supernatants were tested for inhibitory activity. 2.5 Plasminogen, plasmin and urokinase
Plasminogen was isolated by affinity chromatography from FCS according to the procedure of Deutsch and Mertz 1231. The plasminogen eluted with 0.2 M eaminocaproic acid was precipitated overnight with 3.1 g ammonium sulfate/lO ml. After centrifugation (3500 x g, 10 rnin), the precipitate was dissolved in 0.05 M Tris-0.02 M lysine buffer, pH 9.0, [24] at a concentration of 0.2-0.4 CU/ml[25]. Aliquots were stored frozen at -70°C. Plasmin was obtained by activation with urokinase. Urokinase (Serono, Freiburg, FRG) was dissolved in 0.9% NaCl at a concentration of 250 CTA Uiml and stored frozen; 0.1 U was needed to activate 20 p1 plasminogen. This concentration of urokinase contained no fibrinolytic activity.
The caseinolytic assay of plasmin was performed with 14C-labeled N,N-dimethylcasein, prepared as described by Drucker [27]. The hydrolysis of N-p-tosyl-L-arginine methylester (TAME) by trypsin and of N-benzoyl-L-tyrosine ethylester (BTEE) by chymotrypsin were measured by a modification of the esterolytic methods of Hummel [28]. The hydrolysis of H-D-valyl-leucyl-lysyl-p-nitroanilid(S: 2251, which was donated by Kabi, Stockholm, Sweden) by plasmin was measured as follows: to 600 pl D-MEM or inhibitor-containing supernatants Tris-HC1 was added to a concentration of 0.05 M, pH 7.4. The mixture was incubated with 100 pl plasmin (0.25 CU/ml; Kabi) for 5 rnin at 37°C to which 200 y10.875 mM of the substrate was added. The extinction of released paranitroaniline was measured at 405 nm. 2.9 Sephadex chromatography
One hundred to 150 ml of macrophage culture supernatant was centrifuged (25 000 x g, 20 min) and concentrated to 2 mi over an Amicon UM-membrane (exclusion limit 1000 mol. wt.). After a second centrifugation (100000 x g, 30 min), the concentrate was fractionated on Sephadex G-150 columns (1.5 X 120 cm); 0.1 M phosphate buffer containing 0.3 M NaCl was used as eluant. Fractions of 1 ml were collected; 100 pi of
Eur. J. Immunol. 1979.9: 613-619
each fraction was tested for inhibitory activity. The active fractions were pooled and stored frozen at -70°C. 2.10 Isoelectric focusing Isoelectric focusing of the inhibitors was carried out in a sucrose gradient with a micro-preparative apparatus, as described by Sorg and Bloom [29]. At the end of the separation, 100 pl fractions were collected, diluted with 100 pl distilled water and the pH determined in every second sample. To remove the Ampholines (Pharmacia, Uppsala, Sweden), the fractions were dialyzed extensively for 48 h at 4°C against 0.1 M phosphate buffer, pH 7.4, which was frequently changed. The low mol. wt. inhibitor was dialyzed over an Amicon UM 2 membrane.
3 Results
Production of fibrinolysis inhibitors by murine macrophages
615
Table 1. Inhibitory activities of serum-free supernatants from proteose peptone-elicited exudates of single BALB/c mice")
Mouse
No.
Dilutions of macrophage culture supernatants 1:l 1 :2 1:4
48 46 77 34
81 62 43
Inhibition of fibrinolysis (96) 31 36 62 16 13 43 31
24 24 51 5 52 30 1.1
One hundred +I of supernatants were tested with urokinase (2.5 X CU)-activated plasminogen. The fibrinolytic activity of the urokinase-formed plasmin was set at 100%. Each value is the mean of three determinations. Maximal deviation was within f 8%.
3.1 Assay of fibrinolysis inhibitors
v. I The presence of either fibrinolysis inhibitors or plasminogen activators in a culture supernatant can be assayed in the same test, if a mixture of plasmin and plasminogen is used, which by itself causes a lysis of 12sI-labeledfibrinogen up to 50%. When inhibitors are present, the fibrinolytic activity of plasmin will be reduced, whereas plasminogen activator will activate plasminogen and increase the fibrinolysis up to 100%. Plasminogen isolated from FCS by affinity chromatography on lysylSepharose, according to the procedure of Deutsch and Mertz [23], always contains a certain amount of active plasmin and, therefore, can be used for the simultaneous detection of plasminogen activator and fibrinolysis inhibitors. Since it was found (data not shown) that the use of L251-labeledfibrinogen increased the sensitivity of the assay about 4-fold, '251-labeled fibrinogen-coated plates were used throughout without prior 00 conversion to fibrin. In order to detect either plasminogen 25 12.5 6.25 activator or fibrinolysis inhibitors in macrophage culture 1 x 10-3 cu plasminogen supernatants, the following concentrations of plasminogen were used: for the detection of plasminogen activator, a Figure 1. Effects of various macrophage culture supernatants concentration of plasminogen/plasmin was chosen (6 x lo5 cells/cm*) on the fibrinolysis of three different concentrations (2.5 x 10-3CU) [25],which showed no more fibrinolytic activ- of plasmin/plasminogen (C---a). Each point is the mean value of 3 ity, but degraded nearly 100% of fibrinogen after complete determinations. The background fibrinolytic activity was 6%. Peritoconversion of plasminogen to plasmin with either plasminogen neal macrophages: Thio = thioglycollate-induced; LPS = lipopolysacactivator or urokinase. For the detection of fibrinolysis charide-induced; P.P. = proteose peptone-elicited; Cas. = caseininhibitors, the same concentration of plasminogeniplasmin was elicited; W. out = Normal washout. used after conversion of plasminogen to plasmin with urokinase. For the simultaneous detection of plasminogen activators and fibrinolysis inhibitors, a concentration of plasminogeni activator, as described by others [20]. The amount of secreted plasmin was chosen (15 x lop3 CU) which degraded about inhibitory activity of casein or proteose peptone-elicited 50% of the fibrinogen. These concentrations of plasminogeni peritoneal macrophages varied from one experiment to plasmin had to be determined for each batch since the ratio of another. This variation seemed to depend on the physical state of the mice, since the amount of secreted inhibitory activity plasminogen to plasmin was variable. was also different in supernatants of macrophages from individual mice within the same experiment (Table 1). 3.2 Secretion of fibrinolysis inhibitors by peritoneal exudate macrophages 3.3 Chemical characterization of inhibitory activities Inhibitory activity was detected in normal washout and in proteose peptone or casein-elicited macrophages (Fig. 1). LPS- 3.3.1 Sephadex chromatography induced macrophages were not found to secrete inhibitory activity or plasminogen activator. On the other hand, thiogly- Serum-free supernatants of casein and proteose peptone-elicollate-induced macrophages readily produced plasminogen cited macrophages were fractionated on Sephadex G-150.
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C. Sorg
616
Table 2. Recovery of inhibitory activity from a serum-free supernatant of proteose peptone-elicited macrophages after separation on Sephadex G-150
Quantity (ml) 111
Supernatant Inhibitor I Inhibitor I1 Inhibitor 111
125 24 10
Inhibitory activity (%) KCCOVCI!. Dilutions"' ('+) 112 I : J 1 . 8 I : I ~
72'" 77 73 6s
x
56
17
-
-
I00
7x 71 51
6s
5-1 2x
32
44
-
I6
-
-
6
48 20
a) Dilutions were done with elution buffer. b) Mean of three determinations. The fibrinolytic activity of urokinase-activated plasmin was the same as in Fig. 2; mean deviation: k 9%.
Table 3. Inhibition of proteolytic and esterolytic activity of plasmin, trypsin and chymotrypsin by unfractionated supernatants and isolated inhibitor I and I1 a) Sp = unfractionated supernatant. Substrates Plasmin Trypsin bl I = Inhibitor I. sl, II c) I1 = Inhibitor 11. spa' l t ' l I[') sp I I1 d) ++ = completely inhibited. '2'I-labelctl hhrinogcn ++"' + + + + ++ ++ ++ ++ ++ ++ e) n.d. = not done. 'I'AME n.d.'l +'I + t 11.d. f) + = partial inhibition. H'I'EE n.d. n.d. + + g) S : 2251 = H-D-valyl-leucyl-lysyl-I I I s : 225 I P' n.d. n.d. p-nitroanilid. h) - = no inhibiton.
,
Each fraction was assayed for inhibition of the fibrinolytic activity of plasmin. Three activities at the mol. wt. ranges of 60 000, 45 000 and 15 000 were detected (Fig. 2). The recovery of the inhibitory activity applied to the column was about 70% (Table 2). The activity at mol. wt. 60000 was not found in every experiment and was therefore not accessible for extensive characterization. The activity at mol. wt. range 15 000 was hence designated as inhibitor I and at 45000, as inhibitor 11. Supernatants of LPS and thioglycollate-induced macrophages, fractionated and assayed in an identical way, did not contain detectable amounts of inhibitory activity in any fraction (data not shown).
3.3.2 Isoelectric focusing Pooled fractions of the Sephadex G-150 chromatography at the mol. wt. range 15000 (inhibitor I) and 45 000 (inhibitor 11)
were subjected to isoelectric focusing in a sucrose gradient. As shown in Fig. 3 for inhibitor 11, the inhibitory activity peaked at pH 4.1 to 4.3. Isoelectric focusing of inhibitor I (data not shown) gave identical results.
3.3.3 Stability and specificity of inhibitors The activity of both inhibitors was stable during incubation for 1 h at 56 "C and was completely destroyed after incubation for 1 h at 70°C. This, together with the labeling data (Sect. 3.4.) provides evidence that the inhibitors are in major parts of protein nature. In order to determine the sepcificity of inhibitors, supernatants of proteose peptone or casein-elicited macrophages were tested not only in the plasmin but also in the trypsin and chymotrypsin-dependent lysis of fibrinogen (Fig. 4). One hundred p1 of the supernatant obtained from proteose peptone-elicited macrophages, cultivated at a density of 8 X lo5 cells/cm2, caused a 50% inhibition of 0.25 cig chymotrypsin and of 0.3 pg trypsin. When the isolated inhibitors I and I1 were assayed, the proteolytic activity of chymotrypsin and trypsin was similarly inhibited (Table 3). Inhibitor I and % 70
F
In .-
(BSA
il
70
80
(OA
90 100 110 fraction number
-x 50VI
(Chi 120
130
140
150
Figure 2. Sephadex G-150 chromatography of culture supernatants of proteose peptone-elicited peritoneal macrophages. (BSA: bovine serum albumin, mol. wt. 67000; OA: ovalbumin, mol. wt. 45000; Chy: chymotrypsinogen A 25000; Cyt.: Cytochrome C, mol. wt. 12500). Each point is the mean value of three determinations. The fibrinolytic activity of the used urokinase-activated plasmin (2.5 X CU) was 93 rf: 8%.
.-L
e
L
2 4
30
fraction number
Figure 3. Isoelectric focusing (pH 3.5-10) of inhibitor I1 (mol. wt. 45 000) after Sephadex chromatography. The fibrinolytic activity of CU) is shown by the the used urokinase-activated plasmin (2.5 X broken line. pH gradient @---A).
Eur. J. Immunol. 1979. 9: 613-619
Production of fibrinolysis inhibitors by murine macrophages
617
rophages cultured for the first 24 h in the presence of normal FCS contained the highest inhibitory activity. In the other type of supernatants, the inhibitory activity was decreased by about 30-50%. Similar results were obtained when the cell lysates of the corresponding cultures were tested. From these data, it is not clear whether the inhibitory activity represents absorbed and released, or synthesized material. The decrease in inhibitor release by cultures kept either serum-free or with acid-treated FCS might be due to the deteriorating culture conditions or to a gradual change in the plasminogen activatorproducing state induced by the culture conditions. kg trypsin
wg chymotrypsin
Figure 4. Effect of culture supernatants (H from )proteose peptone-elicited macrophages (8 X 10' cells/cm2) on the fibrinolytic activity of trypsin (a) and chymotrypsin (b). Activity of the enzymes alone was measured in D-MEM (H).
11, as well as the unfractionated supernatants, inhibited the esterolysis of TAME by trypsin and of BTEE by chymotrypsin. In this case, however, no complete inhibition could be obtained, neither by the supernatants nor by the isolated inhibitors. In both cases, only 50% of the esterolytic activity was inhibited. If the inhibitors were incubated prior to the experiment for 1 h at 70°C, no inhibition of the esterolytic activity of trypsin and chymotrypsin was observed, which is in line with the experiments on the stability of inhibitors described above. 3.4 Synthesis of inhibitors by macrophages
After the detection and chemical characterization of inhibitory activities in supernatants of certain macrophage cultures, the question was still open whether the inhibitory material was actively synthesized or just released material which had been previously absorbed from the serum in vivo or from the serum in the culture medium. In order to clarify this question, the following experiments were performed. Macrophages were cultivated for the first 24 h in D-MEM containing either 10% FCS, 10% acid-treated, inhibitory-free FCS or 0.2% albumin. All three types of cultures were then transferred to serum-free medium and cultured for another 24 h. When the supernatants were tested for inhibitory activity (Table 4), it was always found that supernatants of mac-
If, however, the inhibitory activity had been previously absorbed from serum either in vivo or from the FCS in vitro, the same inhibitory activities should be detected in normal mouse serum or in FCS, as well as in macrophage culture supernatants after fractionation. Normal mouse serum and FCS were therefore fractionated and tested for inhibitory activity. When the fractions of FCS were tested for inhibition of plasmin, two major peaks of inhibitory activity were found with mol. wts. higher than 80000 (data not shown). Occasionally, a small amount of an additional inhibitory activity with a mol. wt. of 15000-20000 was found which was not dialyzable, in contrast to the inhibitor I from macrophage culture supernatants, and which was lost after a dilution step of 1: 2. No inhibitory activity was ever found at rnol. wts. 25 000-80000. When mouse serum was analyzed the same way, high mol. wt. inhibitors were readily detected but not low mol. wt. inhibitors in the range of 15 000-60 000. These experiments, in connection with the data shown in Table 4, are evidence that the inhibitors are not absorbed from the serum in vivo or from the FCS used in culture medium. Since the experiments described above provided only circumstantial evidence, we investigated whether the inhibitory material could be internally labeled by a radioactive amino acid. Thioglycollate-induced and proteose peptone-elicited macrophages, known to produce either plasminogen activator or inhibitory activity, were therefore labeled with ['HH]leucine. The radiolabeled inhibitory material was detected by specific absorption of the plasmin-inhibitor complex to lysyl-Sepharose. As shown in Table 5 , thioglycollate-induced macrophages do not produce labeled material that binds specifically to plasmin, whereas proteose peptone-elicited macrophages produce material which is specifically bound, amounting to about 6% of the total labeled material which was not found when plasmin was omitted. Experiments performed
Table 4. Inhibitory activities of serum-free supernatants and cell lysates of macrophages after preceding culture in medium containing either normal FCS (n-FCS), inhibitor-free FCS (at-FCS) or no serum
Pcritoncal rnacrophagcs elicited by
Serum uscd in culture
Su pcrna t ant I:l
1::
I:4
C'cll I!\alc 1:l 1.2
Inhihition of (ihrinolvsi\ ( ' i Proteosc peptone
ciwin .
(J.
'
n-FCS at-FCS No," seruni
73
hX
23
sx
56 3x
37 II
Ih
41 33
n-FCS ;lt-FC'S No"' wrum
77 35 73
53
17
15 0
0
II
0
11.d.
(1
)
20 I2 18 I1.ll ."
.d
'
a) The supernatant was tested after 24 h of culture. b) not done.
618
Eur. J. Immunol. 1979. 9: 613-619
V. Klimetzek and C. Sorg
Table 5. Detection of radiolabeled inhibitors by specific binding of plasmin inhibitor complexes to lysyl-Sepharose PcritDneal
Plasmin
macrophages induocdlelicited
supern3tlysylant Srpharose (rpm x fcprn) 10 ’)
by
Tluoglycollatc
’I‘otal Radioac- ci: of rulal radioiictiv- tiviry radioscit); of hound to ti\i;ity
-
1.13
t
1.21
29,168 S1.YKS
0.42
c
I .32 1.33
40.272 81!.Wo‘
0.30 6 1
Proteose peplone
(0.25
to recover the inhibitory activity have as yet been unsuccessful because the plasmin-inhibitor complex could not be dissociated without loss of biochemical activity.
4 Discussion Using the plasmin-dependent lysis of ‘2sI-labeled fibrinogen, it could be shown that nonstimulated washout as well as proteose peptone or casein-elicited macrophages release inhibitors of fibrinolysis, in contrast to thioglycollate-induced peritoneal macrophages which produce plasminogen activator but no inhibitors. The inhibitor production was also not changed after phagocytosis of latex beads (unpublished observations). Macrophages from LPS-induced peritoneal exudate cells have been described to release plasminogen activator only after phagocytosis of latex beads [30]. Inhibitors were also missed in culture supernatants of LPS-induced macrophages before and after phagocytosis of latex beads. Inhibitors and plasminogen activator have so far not been observed to be produced simultaneously by cells of the same culture. The possibility that both activities are produced and that the differences are quantitaive in nature could be ruled out by experiments where the supernatants were fractionated on Sephadex. Plasminogen activator and inhibitors which have different mol. wts. were not detected in the same culture supernatants. By Sephadex chromatography, the mol. wt. of inhibitors was determined at 60000, 45 000 and 15 000, which clearly shows that the inhibitors are different from az-macroglobulin which has been described to be produced by human rnonocytes [311. The inhibitors were found to block the proteolytic and esterolytic activity of trypsin and chymotrypsin. On the other hand, only the proteolytic activity of plasmin was inhibited. Whether the inhibitors can also inhibit plasminogen activator is not known at present, since the determination of the split product of plasminogen requires different methods [32]. It certainly would be most interesting to see whether inhibitor-producing macrophages are the direct antagonists of the plasminogen activator-producing rnacrophages. An important question was whether the inhibitors are actively synthesized or whether they are material which had been taken UP by macrophages from the Serum in viuO 01‘from the serum added to the culture medium. Evidence for an active synthesis came from the chemical characteristics of the
inhibitors which are different compared to all known serum protease inhibitors (331. Along the same lines, it was shown that mouse serum did not contain any detectable amounts of low-mol. wt. inhibitors. Peritoneal macrophages which were cultured in serum-free medium nevertheless released inhibitors of lower molecular size. On the other hand, the possibility exists that macrophages take up high-mol. wt. inhibitors which are subsequently released in degraded though active form. Strong evidence for a synthesis of inhibitors came from experiments using radioactive leucine for internal labeling of inhibitors. The plasmin-inhibitor complexes were detected by specific absorption to lysyl-Sepharose. The amount of inhibitors produced is quite substantial with 6% of the total macromolecular counts in the culture supernatants. Since the absorbed labeled material could not be desorbed for further functional and chemical characterizations, the final proof for identity of labeled material and of inhibitory activity could not be achieved. The possibility that the labeled material is just a substrate for plasmin seems unlikely because of the negative control experiment performed with supernatants from thioglycollate-induced macrophages. From these data, it appears that the release of fibrinolysis inhibitors is a property of macrophages in a certain functional state. Others have reported a wide variety of functions which are enhanced or newly acquired by macrophages after exposure to lymphokines [7,11-15,261. An interesting question, therefore, would be whether the production of fibrinolysis inhibitors is modulated under the influence of lymphokines. In recent experiments (V. Klimetzek and C. Sorg, in preparation) it was found that inhibitor production by macrophages is reduced or even completely abolished after exposure to lymphokines from mitogen-stimulated spleen cells. Cultures of casein or proteose peptone-elicited macrophages could even be converted from the inhibitor to the plasminogen activatorproducing state. Whether the release of plasminogen activator is a function of “activated” macrophages or a constitutive property of macrophages in a certain maturation state, is currently being investigated. Studying the differentiation of bone marrow cells into macrophages in a liquid culture system, evidence has been obtained suggesting that plasminogen activator-producing macrophages can switch to the inhibitorproducing state in the course of differentiation [34]. W e thank Mr. Erhard Kreinjobst and Ms.Brigitte Brokmeier for expert technical help. Received February 24, 197s; in revised form March 23, 1979.
5 References 1 Van Furth, R. (Ed.), Mononuclear Phagocytes in Immunity, Infection and Pathology, Blackwell Scientific Publications. Oxford 1975. 2 Mackaness, G. B., in Mudd, S. (Ed.), Infectious Antigens and Host Reactions, Saunders, Philadelphia 1970, p. 61. 3 Krahenbuhl, J . and Remington, I. S . , Infect. Immun. 1972.4: 337. 4 Rosenthal, A. S . , Blake, J. T., Ellner, J. J . , Greineder, D. K. and Lipsky, P. E., in Nelson, D . S. (Ed.), Immunobiology of the Macrophage, Academic Press, New York 1976, p. 131. 5 Rosenstreich, D. L. and Oppenheim, J. J., in Nelson, D. S. (Ed.), Immunobiology of the Macrophage, Academic Press, New York 1976, p, 162, 6 Gery, J., Gershon, R. K. and Waksman, B. H., J . Exp. Med. 1972. 136: 128. 7 Neumann, C. and Sorg, C., Eur. J . Immunol. 1977. 7: 719.
Eur. J. Immunol. 1979.9: 619-625
Lipopolysaccharide and lipid A-induced human B cell activation
8 Nelson, D. S . , in Nelson, D. S. (Ed.), Immunobiology ofthe Macrophage, Academic Press, New York 1976, p. 235. 9 David, J. R., Fed. Proc. 1975. 34: 1730. 10 Walker, W. S., in Nelson, D. S . (Ed.), lmmunobiology of the Macrophage, Academic Press, New York 1976, p. 91. 11 Remold-O'Donnell, E . and Remold, H. G., J . Biol. Chem. 1974. 249: 3622. 12 Hammond, N. E. and Dvorak, H. S., J . Exp. Med. 1972. 136: 1518. 13 Wahl, L. M., Wahl, S. M., Mergenhagen, S. E. and Martin, G. R., Science 1975. 187: 261. 14 Klimetzek, V. and Sorg, C., Cell. Immunol. 1976. 27: 350. 15 Vassalli, J. D. and Reich, E., J . Exp. Med. 1977. 145: 429. 16 Karnovsky, M. L., Lazdins, J., Drath, D. and Harper, A,, Ann. N Y Acad. Sci. 1975. 256: 266. 17 Allison, A. C. and Davies, P., in van Furth, R. (Ed.), Mononuclear Phagocytes in Immunity, Infection and Pathology, Blackwell Scientific Publications, Oxford 1975, p. 487. 18 Pantalone, R. N. and Page, R. C., Proc. Nut. Acad. Sci. USA 1975. 72: 2091. 19 Nathan, C. F., Karnovsky, M. L. and David, J. R., J . Exp. Med. 1971. 133: 1356. 20 Unkeless, J . C., Gordon, S. and Reich, E., J . Exp. Med. 1974. 139: 834.
C. I. Edvard Smitho, Lennart Hammarstrom', A. Graham BirdoA, Takao Kunori ,Bjorn Gustafsson' and Tord Holme'
Department of Immunobiology, Wallenberglaboratory', Department of Bacteriology', Karolinska Institute, Stockholm and Transplantation Immunology Laboratory, Huddinge Hospital, Huddinge'
21 Newcomb, E. W., Silverstein, S. C. and Silagi, S . , J . Cell. Physiol. 1978.95: 169. 22 Unkeless, J. C . , Tobia, A . , Ossowski, L., Quigley, J. P., Rifkin, D. B. and Reich, E., J . Exp. Med. 1973. 137: 85. 23 Deutsch, D. G . and Mertz, E . T., Science 1970. 170: 1095. 24 Summaria, L., Arzadon, L., Bernabe, P. and Robbins, K. C., J , Biol. Chem. 1972. 247: 4691. 25 Remmert, L. F. and Cohen, P. P., J . Biol. Chem. 1949. 181: 431. 26 Klimetzek, V. and Sorg, C., Eur. J . Immunol. 1977. 7: 185. 27 Drucker, H., Anal. Biochem. 1972. 46: 598. 28 Hummel, B. C. W., Can. J . Biochem. Physiol. 1959. 37: 1393. 29 Sorg, C. and Bloom, B. R . , J . Exp. Med. 1973. 137: 148. 30 Gordon, S., Unkeless, J. C. and Cohn, Z. A., J . Exp. Med. 1974. 140: 995. 31 Hovi, T., Mosker, D. and Vaheri, A , , J . Exp. Med. 1977. 145: 1580. 32 Dano, K. and Reich, E . , in Reich E., Rifkin, D. B. and Shaw, E. (Eds.), Proteases and Biological Control, Cold Spring Harbor Conferences on Cell Proliferation 1975, vol. 2, p. 357. 33 Wiman, B. and Collen, D., Nature 1978. 272: 549. 34 Sorg, C., Neumann, C., Klimetzek, V. and Hannich, D., in van Furth, R . (Ed.), Mononuclear Phagocytes - Functionaf Aspects, Martinus Nijhoff Medical Division, The Hague, Boston, London 1979, in press.
Lipopolysaccharide and lipid A-induced human blood lymphocyte activation as detected by a protein A plaque assay* Various purified cell wall lipopolysaccharides (LPS) from gram-negative bacteria and derivatives of these LPS were tested for their stimulatory capacity for human peripheral blood cells. Immunoglobulin (Ig) production was tested by an indirect plaque-forming cell assay using Staphylococcus aureus protein A-coupled erythrocytes and specific anti-Ig as developing serum. This method allows the detection of the majority of cells secreting Ig of a single class, and the number of plaque-forming cells detected are approximately 10CL1000 times the amount obtained using normal sheep red cells as targets. LPS containing the 0 antigen-specific chain, as well as mutant products only containing lipid A and ketodeoxyoctonate trisaccharide, could induce cell division and antibody synthesis. The polypeptide antibiotic polymyxin B was found to inhibit LPS-induced activation. Furthermore, purified lipid A, comulexed with bovine serum albumin, was also found to activate human peripheral blood B cells. These findings demonstrate that human peripheral blood lymphocytes can be activated by LPS and also indicate that lipid A is the active part of these molecules. [I 24101
* This work was supported by the Swedish Cancer Society and the A
619
Torsten and Ragnar Soderberg Foundation. Radcliffe Travelling Fellow, Oxford University, GB.
Correspondence: C. I. Edvard Smith, Department of Immunobiology, Wallenberglaboratory, Lilla Frescati, S-104 05 Stockholm 50, Sweden Abbreviations: BSA: Bovine serum albumin BSS: Balanced salt solution LPS: Lipopolysaccharide PFC: Plaque-forming cell(s) PPD: Purified protein derivative of tuberculin PWM: Pokeweed mitogen [jH]dThd: Tritiated thymidine PBL: Peripheral blood lymphocytes SRBC: Sheep red blood cells 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979
1 Introduction Lipopolysaccharides (LPS) have been extensively used in experimental immunology due to their lymphocyte-activating properties [l]. These mitogens have been shown to activate resting mouse B lymphocytes to proliferation, maturation and immunoglobulin (Ig) secretion in a polyclonal fashion [ 2 ] and have thus been designated polyclonal B cell activators. Approximately one-third of all mouse splenocytes are reactive to LPS [3], and the ability to respond is dependent on a gene allocated on chromosome 4 [4]. In the large majority of reports dealing with LPS-induced activation, the effect on mouse splenocytes has been studied. 00l4-2980/79/0808-0619$02.50/0
