LEUKOCYTE INHIBITORY FACTOR ACTIVATES HUMAN NEUTROPHILS AND MACROPHAGES TO RELEASE LEUKOTRIENE B4 AND THROMBOXANES Pio Conti, Marcella Reale, Renato C. Barbacane, Maw-o Bongrazio, Maria R. Panara, Stefano Fiore, Roy Dempsey*, Larry Borish** Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalamine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of >95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury. LIF may have an important function as a PMN and macrophage activator. Our observations demonstrate the inflammatory potential of LIF and extend the range of activities of this lymphokine to PMNs and macrophages.

o 1990 by W.B. Saunders Company.

Human polymorphonuclears (PMNs) and macrophages generate prostaglandins, thromboxanes, and leukotrienes, in response to a variety of soluble or particulate receptor-mediated stimuli.lM3 Cytokines were recently shown to stimulate the production of arachidonic acid oxygenation products, which were derived from both cyclooxygenase and lipoxygenase-catalyzed path-

Immunology Division, Institute of Experimental Medicine, University of Chieti, Via dei Vestini, 66100 Chieti, Italy; *Endogen, Boston, MA; **Allergy Division, Department of Medicine, New England Medical Center, Boston, MA. To whom reprint requests should be addressed. o 1990 by W.B. Saunders Company. 1043/90/0202-0006%05.00/0 KEY WORDS: boxanes/Mediation

142

Leukocyte

inhibitory

factor/Leukotriene

B4/Throm-

ways. 4-gThe in vivo thromboxane synthesis is associated with many pathophysiologic phenomenas including thrombocytopenia, intravascular coagulation, pulmonary and mesenteric vasoconstriction, and endotoxic shock.10-12 In fact, experimental immunotherapy with certain cytokines can induce several hematologic and metabolic changes typical of host response to infection and injury, such as hypotension, decreased systemic arterial pressure, thrombocytopenia, leukopenia, edema, and hemorrhage.13 These effects could be mediated by the Slipoxygenase product, leukotriene B4 (LTB4). This leukocyte product mediates inflammation by producing cell aggregation, lysosomal enzyme release, and chemotaxis.‘4-15 LTB4 metabolites possess much less biological activity.16-17 The cytokine, leukocyte inhibitory factor (LIF), is a lymphocyte product with a molecular weight of 58,000, CYTOKINE,

Vol. 2, No. 2 (March),

1990: pp 142-148

activationof neutrophils /

LIH

which exerts multiple biological activities.18 It has been reported recently that LIF directly induces specific granule secretion in PMNs, stimulates neutrophilendothelial cell adhesion,lg and potentiates a number of formyl-methionyl-leucyl-phenylalanine (FMLP)-mediated functions such as aggregation and cell activation. It is well known that cytokines are potent stimulators of prostaglandins and thromboxanes from many different cells*‘-*’ and when human neutrophils are exposed to appropriate stimuli they will aggregate22-23 and generate thromboxanes24-25 and leukotrienes.26 Since arachidonic acid products (i.e., LTB4 and TxA2) are very potent aggregating, chemokinetic, and vasoconstrictive substances, we have tested the ability of LIF, along with other cytokines, to stimulate the arachidonic acid cascade of human PMNs and macrophages. In our experiments the production of LTB4 and TxA2 (detected as TxB2) were strongly stimulated by LIF. On the other hand, interleukin 6 (IL 6) and granulocyte-monocyte colony stimulating factor (GM-CSF), both failed to produce stimulation of LTB4 and TxA2 by PMNs.

RESULTS Using the radioimmunoassay method, the generation of LTB4 and thromboxane by PMNs and macrophages was studied following treatment with LIF, IL 6, and GM-CSF. After pretreatment with cytochalasin B neutrophils (5 x 106/ml) and mono(W (5 e/ml), cytes (5 x 106/ml) from peripheral blood were exposed to A23 187 and FMLP. A23187 (5 PM), FMLP (5 x lo-’ M), and LIF were added at different concentrations to the cultures, generating a significant increase of LTB4 and thromboxanes, when compared with the controls. Treatment with IL 6 and GM-CSF did not produce significant increments of these two arachidonic acid products. In Table 1, we show a time course study where LIF (1 U/ml) notably stimulated the 5-lipoxygenase products LTB4 by PMNs after incubating for 5 min and continued stimulation for 30 min. However, the release of LTB4 after 30 min incubation with LIF was less than the release at 5 and 15 min, (probably because the neosynthesized LTB4 is metabolized via w-oxidation). TABLE

1.

Time course production

No significant increase of LTB4 was found at 0 time, which was made directly in ice, or at 1 min incubation at 37°C. When PMNs were treated with A23187 (5 PM) similar effects were obtained, but the release of LTB4 was much higher than those obtained with LIF. FMLP also increases LTB4 production by PMNs significantly after 5, 15, and 30 min. Figure 1 shows treatment of PMNs with LIF at 4,2, 1, and 0.5 U/ml, after CB (5 pg/ml) resulted in a dose-dependent increase of LTB4 that was 472.5 * 95.4, 337.0 * 95.0, 285.7 ? 22.0, and 167.2 + 41.3 pg/ml, respectively. PMNs treated with LIF at 0.25 U/ml did not significantly enhance LTB4 production (48.3 + 8.0 pg/ml) when compared with the control (33.3 + 17 pg/ml). When the cells were treated with A23187 (5 PM), a greater increase of LTB4 was found (4710 i 42.4) as compared with LIF. LTB4 production by PMNs, after 15 min incubation, is shown, following treatment with A231 87, GMCSF (200 U/ml), IL 6 (500 U/ml), and LIF (1 U/ml) (Table 2). The results show that LIF, but not GM-CSF or IL 6, enhances LTB4 production when compared with the controls. A23 187 treatment produced a 20-fold increase in LTB4 release, compared with LIF. Table 3 is a time course study of TxA2, detected as TxB2, production by PMNs. The cells were treated with A23187 (5 PM), FMLP (5 x lo-’ M), and LIF (1 U/ml). We demonstrate that LIF and FMLP can stimulate the cyclooxygenase products from 1 min to 30 min incubation. A23187 induced additional release of TxA2. In these experiments, the generation of TxA2, detected as TxB2, could not be attributed to platelet contamination of the PMN suspension. The quantity of platelet contamination (10: 1 PMN) to the cell cultures was not sufficient to influence the amount of TxB2 measured in the reaction mixtures containing either resting cells or stimulated cells with A23187 or FMLP. Table 4 shows TxA2 production by PMNs in a dose-dependent curve. LIF used at 4, 2, 1, 0.5, and 0.25 U/ml gave a dose-dependent increase of TxA2 release that was 189 + 10.1, 158.0 + 12.0, 120.0 + 18.5,79.0 +16.0, and 43.0 k 14.4 pg/ml, respectively. In addition, A23 187 (5 PM) provoked a strong release of TxA2. This

of LTB4 by PMNs PMNs

Treatment

0 Time

Control A23187 FMLP

LIF

(5 x IO-’

(1 U/ml)

M)

143

49.5 + 14.0 39.0 t 16.9 61.5 + 14.8 42.9 + 21.2

1 min

24.0 36.0 29.2 22.2

f + + i-

LTB4

Production 5 min

4.0 8.0 7.0 3.3

85 4725 108 375

+ 19 f 142 ZL 46 + 21.2

+ SD (pg/ml) 15 min

162 4570 2985 427.5

+ k + zt

63.6 318 233 10

30 min

136 3450 1070 298.0

-e 6.0 t 424 i 180 + 23

Time course experiment with natural purified LIF (1 U/ml), A23187 (5 PM) or FMLP (5 x lo-’ M) on LTB4 production. Times after incubation with either culture media alone (control) or culture media containing LIF (1 U/ml), A23187 (5 rM) or FMLP (5 x 10e7 M). The mean and standard deviation of LTB4 concentration in culture media are displayed for each time of treatment.

144 / Conti et al.

CYTOKINE,

Is3 H23187 5 ur1 was higher than LIF (502 + 41.1 pg/ml) when compared with the control (24 f 11 .O pg/ml). Macrophages treated with LIF (1 U/ml) also caused a significant release of TxA2. The treatment of cells with A23 187 (5 PM) caused a more significant generation of TxA2, compared with LIF (Fig. 2). The release of LTB4 by macrophage after treatment with different concentrations of LIF is shown in Table 5. The effect of LIF on generating LTB4 was dose-dependent. LTB4 release by macrophages following treatment with A23 187 (5 yM) was about 15- to 20-fold more than treatment with LIF alone. Figure 3 shows the role played by leukocyte inhibitory factor in activating PMNs during an inflammatory reaction. The stimulation of arachidonic acid products (LTB4 and TxA2) by LIF confirms the important role played by LIF as a proinflammatory cytokine.

TABLE 2. Production after 15 min incubation

of LTB4 by PMNs

15 min incub.

LTB4 production (pg/mO 2 SD

PMNs Control A23187 (5 PM) GM-CSF (200 U/ml) IL-6 (500 U/ml) LIF (1 U/ml)

82.5

6150

f

14

+ 63.6

88.0 87.0

k 60 + 28

315.0

+ 67

Effect of purified LIF (1 U/ml), IL-6 (500 U/ml), GM-CSF (ZOO U/ml), and A23187 (5 MM) on the production of LTB4 by PMNs (5 x 106/ml) in culture. The mean and standard

deviation

are displayed

for each treatment.

Vol. 2, No. 2 (March

1990: 142-148)

Figure 1. Effect of natural purified LIF or A23187 (5 uM) on LTB4 production by PMNs in culture for 15 min. A dose-dependent stimulation is observed. The mean and standard deviation of LTB4 concentration in culture media arc displayed for each dose of LIF.

DISCUSSION Human PMNs are cells that participate in many pathophysiological events. It has been demonstrated recently that PMNs have LIF receptors.27‘2g In this study, we have used human PMN leukocytes and demonstrated the ability of the lymphokine LIF to enhance LTB4 and TxA2 production by these cells. LTB4 (5 to 12 diHETE) is a potent chemokinetic, vasoconstrictive, and proaggregatory substance that plays an important role in the activation of PMNs and inflammation.14,30 LTB4 and TxA2 may also contribute to disseminated intravascular coagulation and pulmonary hypertensive sequelae of endotoxic shock.13 It has been reported that a number of lymphokines have potent biological activities. This is shown particularly in their ability to act directly on the arachidonic acid cascade, for example, endogenous pyrogen, (IL l), which can provoke the release of arachidonic acid metabolites that cause fever and mediate infection, injury, and immunological reactions.31 Recently Dinarello34 emphasized that cytokines can act to promote aggregation and stimulate thromboxane formation by neutrophils. This cannot be considered only a laboratory observation since it has been confirmed in human body fluids. These include in vivo inflammatory joint fluids. 34 Recent evidence suggests that LIF, a lymphokine released by T-cells, modulates several FMLPinduced neutrophilic functions, such as enhancing aggregation following exposure to calcium ionophore A23 187.2g,33We demonstrate in this study the ability of

LIH

TABLE

3.

Time course production

Control A23187 (5 fiM) FMLP (5 x lo-’ LIF (1 U/ml)

0 Time

M)

18.7 36.5 21.5 28.0

* t + +

1 min

10.2 30.4 3.5 10

38.5 35.0 43.5 150.0

* + + i

2.1 4.2 6.3 10

LIF to enhance PMN and macrophage production of LTB4 and TxA2. This extends further our prior results, which indicated that LIF may have an important function as a macrophage activator.34 We have also found that other cytokines, which tested GM-CSF or IL 6, did not enhance LTB4 production by PMNs or macrophages. In fact, GM-CSF, which possesses many biological activities, fails to produce fever when the recombinant form is injected. This effect may be explained by the fact that it does not stimulate arachidonate products.21 LIF’s ability to activate PMN and macrophage production of 5-lipoxygenase and cyclooxygenase products is in accordance with other studies. These indicate the capacity of LIF to mediate PMN and macrophage functions and, therefore, may play a central role in inflammation.2g-33 Since other cytokines have been shown to induce in vivo hypotension, systemic vascular resistance, thrombocytopenia, and leukopenia, which are all involved in the toxic shock syndromes, LIF can affect PMNs and macrophages possibly by the same mechanism of stimulating arachidonic acid metabolites. Our results may imply an important mechanism for death by sepsis, since it has been reported previously that cytokines induce the release of arachidonic acid metabolites by neutrophils and macrophages, and through stimulation of endothelial cell-leukocyte adhesion.13

TABLE 4. Dose response values of TxB2 after PMNs treatment with LIF

Control A23187 (51~ M) LIF 4 U/ml LIF 2 U/ml LIF 1 U/ml LIF 0.5 U/ml LIF 0.25 U/ml

PMNs (5 x 106/ml) TxB2 (pg/ml) f SD

24 502 189 158 120 79 43

f -t f f f k +

(pg/ml)

5 min

Time course experiment with natural purified LIF (1 U/ml), A23 187 (5 pM), or FMLP either cell culture media alone (Control) or cell culture media containing LIF (1 U/ml), TxB2 concentration in culture media are displayed for each time of treatment.

Treatment

of neutrophils

/

145

of TxB2 by PMNs PMNs TxB2 production

Treatment

activation

11.0 41.1 10.1 12.0 18.5 16.0 14.4

Effect of natural purified LIF at various concentrations and A23187 (5 PM) on TxB2 production by PMNs in culture for 15 min. The mean and standard deviation of the TxB2 concentration in culture media are displayed for each treatment.

38 360 175 170

f + i +

2.1 98.9 63.6 7.0

k SD 15 min 56.2 960 121 237

f + f +

3.5 381 41.0 60

30 min 71 955 160 335

t ii f

10 134 28.2 70

(5 x lo-’ M). Supernatants were collected at various times after incubation with A23 187 (5 MM) or FMLP (5 x lo-’ M). The mean and standard deviation of the

The demonstrated effect of purified LIF on LTB4 biosynthesis, by both human PMNs and macrophages, cannot be attributed to contamination by endotoxins. We know this because nanogram quantities of bacterial endotoxin are required to elicit such an effect in human PMNs and macrophages. To conclude, we provide new evidence of the stimulatory potential of LIF on enhancing the arachidonic acid metabolites, LTB4 and thromboxanes by PMNs and macrophages, demonstrating further the inflammatory potential of LIF and further extending the range of activities of this lymphokine to PMNs and macrophages. These results show that LIF could serve as a biological signal for PMN and macrophage activation in inflammatory sites and immunological reactions in vivo.

MATERIALS AND METHODS Blood was obtained from healthy donors and collected in polypropylene tubes containing sufficient heparin to obtain a final concentration of 20 U/ml. Tests were performed immediately after collection. The whole blood was collected on Ficoll-Hypaque solution and centrifuged at 400 g for 30 min as described.35 Mononuclear cells were recovered at the interface and were washed three times with HEPES-buffered (10 mM) Hank’s balanced salt solution (HBSS) and reSuspended at the desired concentration in RPM1 1640 medium (GIBCO), supplemented with 10% heat inactivated fetal bovine serum, L-glutamine, and HEPES. Aliquots of 2.5 ml were seeded into petri dishes and incubated at 37”C, 5% CO,-humidified atmosphere for 60 min. After that time, the supernatants, which contained nonadherent cells, were discarded and the remaining adherent macrophages (about 10% of the original population) were recovered by vigorous washings and scraping of the plates. They were then collected in a 30-ml polypropylene tube (Falcon), washed, and cultured in phosphate buffered saline (PBS) buffer for radioimmunoassay. PMNs were isolated as shown.35 After washing the cells three times with buffered saline, the red cells were lysed hypotonically. The isolated cells were washed and resuspended in PBS buffer and their viability examined by trypan blueexclusion method. The cell suspension consisted of 95% PMN leukocytes. The platelet concentration in the PMN suspensions were not high enough to influence the test results (data not shown). Prior to culture, the cells were counted in a Burker

CYTOKINE,

146 / Conti et al.

Vol. 2, No. 2 (March 1990: 142-148)

Figure 2. Effect of natural purified LIF (1 U/ml) and A23187 (5 uM) on TxB2 released by macrophages (5 x 106/ml) after 15 min. incubation. The mean and standard deviation of the TxB2 concentration in culture media are displayed.

counting chamber and the preparation had a PMN to platelet ratio of 10: 1 or less. Samples were treated with LIF, IL 6, and GM-CSF at different concentrations. The controls received a vehicle, at the same concentrations, employed with the test agents A23 187, FMLP, or lymphokines, and radioimmunoassay was determined.

Cytochalasin

B

All macrophage and PMN tests were preincubated with CB (5 pg/ml) for 15 min at 37“C in a shaking water bath before adding the stimuli. CB was dissolved in DMSO, resulting in a final concentration of 0.1% vol/vol, which had no influence on the test results.

Tested Drugs Calcium ionophore A23187 was dissolved in dimethyl sulfoxide (DMSO). Placed in glass cuvettes for each experiment, cells were exposed only to the vehicle at the identical concentrations, in order to determine nonspecific effect. Results are expressed in pg/ml. With each donor, each test and all controls were done in triplicates. FMLP was dissolved in DMSO and further dilutions were made in PBS medium. Incubations were followed by the determination of TxB2 levels.

TABLE 5. Dose response values of LTB4 production macrophages treated with LIF

Treatment

Control (5 PM) (4 U/ml) (2 U/ml) (1 U/ml) (0.5 U/ml)

A23187

LIF LIF LIF LIF

LIF was generated from phytohemagglutinin (PHA) stimulation of peripheral blood mononuclear cells purified 80,000-fold to apparent homogeneity and with an apparent molecular weight of 58,000 by the Endogen Inc. (Boston, MA), following the published method of Bendzten et a1.36,3g GM-CSF and IL 6 were kindly provided by Endogen and the dilutions were made in PBS medium and used at 200 and 500 U/ml, respectively.

by

Radioimmunoassay

M@ (5 x 106/ml) LTB4 production (pg/mI) * SD

32 5840 386 325 289

Cytokines

+ k 5 L f

20.0 401.9 28.7 51.2 18.9

106 i 23.3

Effect of natural purified LIF at various concentrations and A23187 (5 PM) on LTB4 production by macrophages (5 x 106/ml) after 15 min incubations. The mean and standard deviation of the LTB4 concentration in culture media are displayed for each treatment.

of LTB4 and TxB2

After the purification, as previously described, macrophages (5 x 106/ml) were suspended in RPM1 1640 (without any serum) and dispensed into polypropylene tubes. The cell incubations, either with or without cytokines or A23187 or FMLP, were added to the cell suspensions at different concentrations. All the controls received DMSO at the same concentration as the calcium ionophore A23187 or FMLP. Following centrifugation of the incubation mixture, the cell-free supernatants were stored at -70°C before radioimmunoassay.37,38 Radioimmunoassay of LTB4 and TxB2 were determined in pg/ml. Each test and all controls were done in triplicate.

LIH

ENHANCED CHEMOTAXIS

activation of neutrophils / 147

SUPEROXIDE

RESPIRATORY BURST

Figure 3. Biological activity of leukocyte inhibitory factor.

Materials The following

materials used in our study are listed below.

Heparin (20 U/ml), PBS, and trypan blue (Biochrome KG, Berlin, FRG) Ficoll-Hypaque solution (1.007 g/ml), HEPES-buffer (10 mM), calcium ionophore A23187 (5 PM), FMLP (5 x lo-’ M), CB (5 pug/ml), and DMSO (Sigma, St. Louis, MO) RPM1 1640 medium LIF, IL 6, and GM-CSF (ENDOGEN) Standard TxB2 and antibodies against TxB2 and LTB4 (Advanced Magnetic, Boston, MA) 3H-LTB4 (spec. act. 25 Ci/mM) and 3H-TxB2 (specific activity 111.4 Ci/mM) (New England Nuclear, Boston, MA) Standard LTB4 was a kind gift from Dr. J. Rokach (Merck Frosst, Canada) HBSS, fetal bovine serum (lo%), and L-glutamine (0.3 mg/ml) (FLOW Laboratories, Milan, Italy)

Acknowledgments We wish to acknowledge Dr. C.A. Dinarello for his help and suggestions. This study was supported in part by the Italian Ministry of Education. We also thank the AVIS blood donor group of Pescara, Italy.

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