JOURNAL OF PATHOLOGY, VOL.
165 319-323 (1991)
ALPHA-1 ANTICHYMOTRYPSIN IS INCREASED IN HUMAN ALVEOLAR MACROPHAGES BY PHORBOL MYRISTATE ACETATE OR LIPOPOLYSACCHARIDE A N D RELEASED FROM THESE ACTIVATED MACROPHAGES BY GLUCOCORTICOID TOMOFUMI NAGAREDA, MASASHI TAKEDA, KAYO KOJIMA, AKIRA TANAKA, NOBUYUKI TERADA, TOMOYOSHI YAMASAKI, TAEKO NAGAREDA, HIROSHI UENO A N D KIYOSHI KOTOH
Department of Pathology, Osaka University Medical School, Fukushima-ku, Osaka, 553; Department of Pathology. Osaka Medical College, Takatsuki. Osaka, 569, Japan Received 9 October 1990 Accepted 13 June 1991
SUMMARY Alveolar macrophages were obtained from 23 patients and the effects of phorbol myristate acetate (PMA),lipopolysaccharide (LPS), and dexamethasone (DEX) on the proportion of cells with intracellular alpha-1 antichymotrypsin (ACT), and concentrations of ACT in the culture medium were studied in vitro. The alveolar macrophages were obtained by bronchoalveolar lavage at autopsy or from resected lungs at operation and were cultured in suspension for 3 days in medium containing PMA, LPS, DEX, PMA+DEX, or LPS+DEX. Both PMA and LPS significantly increased the percentage of macrophages with intracellularACT. Dexamethasone did not increase the number of ACTpositive cells and significantly suppressed the increaseinduced by PMA or LPS, releasing ACT into the culture medium. The release of ACT from macrophages may contribute to the anti-inflammatoryeffects of corticoids. KEY WORDS-Human
alveolar macrophage, alpha-1 antichymotrypsin,phorbol myristate acetate, LPS glucocorticoid.
INTRODUCTION Alveolar macrophages play a major role in pulmonary defence. The actions of some macrophages require direct cell-to-cell interaction, whereas those of others are mediated through secretion of various substances such as prosta landin,’ complement component,2growth factors,K.~nt e rfe ron,~ and interleukin 1.5 Alveolar macrophages can also synthesize and secrete alpha-] antichymotrypsin (ACT),6 which is a glycoprotein that increases in amount in the acute phase of inflammati~n.’~~ Pulmonary inflammation is induced experimentally by phorbol myristate acetate (PMA) or lipopolysaccharide (LPS): PMA induces interstitial pneumonitis’ while LPS induces pneumonia.” Addressee for correspondence: Dr Tomofumi Nagareda, Oomino 91-36, Sakai, Osaka, 588, Japan.
0022-34 1 719 1 / I 203 19-05 $05.00 0 1991 by John Wiley & Sons, Ltd.
Glucocorticoids suppress inflammation of the lung.” In the present study, we investigated the in vitro effects of PMA, LPS, and glucocorticoid on the proportion of human alveolar macrophages with intracellular ACT and the concentration of ACT in the culture medium. Intracellular ACT was evaluated by immunocytochemical staining of ACT. MATERIALS AND METHODS
Collection of bronchoulveolurluvugejuid Human alveolar macrophages were obtained by bronchoalveolar lavage of 21 right lungs at autopsy and of a right and a left lobe removed surgically from two patients, both with squarnous cell carcinoma. These 23 patients ranged from 40 to 82 years old. The primary diseases of the autopsy cases were
320
T. NAGAREDA ET AL.
hepatocellular carcinoma with liver cirrhosis (7 cases), myocardial i n f a r ~ t i o ndiabetes ,~ mellitus (3), gastric cancer (2), renal failure (I), metastatic brain tumour from breast cancer (I), peritonitis (I), pancreatic cancer (I), laryngeal cancer (I), and cryptococcosis. (1) The pathological findings in the lungs were focal pneumonia in 10 cases, pulmonary oedema in 8, emphysema in 2, metastatic carcinoma in 2, and congestion in 1. Lavage was carried out ten times by instillation of sterile saline (5 ml) into bronchi S' or S3 in autopsy cases within 13 h after death and SIo in the operation cases, and gentle aspiration of fluid was carried out with a syringe. These bronchi and segments were all free from severe pulmonary lesions. Culture of macrophages in experiments I and I1 The bronchoalveolar lavage fluid was obtained from patients 1-7 in experiment I and from patients 8-14 in experiment IT. The lavage fluid was centrifuged at 500g for 5 min, and the cell pellet was washed twice with 5 ml of RPMI-1640 medium supplemented with 10 per cent fetal calf serum (FCS) and finally suspended in 4ml of RPMI-1640 medium with 10 per cent FCS. Samples of 1 ml of the cell suspension were introduced into individual plastic tubes with 4 ml of RPMI-I 640 medium containing 10 per cent FCS with or without PMA (Consolidated Midland Corp., Katonah, NY), LPS (Paesel GmbH, Germany), dexamethasone (DEX), PMA + DEX, or LPS DEX, and the macrophages was finally cultured in 5 ml of medium. The concentrations (in pg/ml) of PMA, LPS, and DEX were, respectively, 0.2, 10, and 0.04 in experiment I; and 0.1, 5 and 0.02 in experiment 11. The final concentrations of the agents in experiment I1 were half of those in experiment I. Macrophages were cultured in suspension for 3 days under 5 per cent CO, in air at 37°C. Then, 2Opl of Indian ink was introduced into each tube, and 15 min later the cells were collected by centrifugation at 500g for 5 min and the cell pellet was washed twice with phosphatebuffered saline (PBS, pH 7.2). In experiment I, cells were smeared on a slide glass and in experiment 11, a cell block was made by adding 40p1 of human serum, 2 W O pl of 0.025 mM calcium chloride, and 2 0 4 0 pl of 200 p/ml thrombin.
+
Culture of macrophages in experiments Illand IV The lavage fluid was obtained from patients 1518 in experiment I11 and from patients 19-23 in experiment IV. Cells in the lavage fluid were collected as described above. The cell suspension in
PBS was transferred into an uncoated plastic culture dish and incubated in 5 per cent CO, air at 37°C for 30 min. Then the medium was removed and macrophages attached to the dish well were washed with PBS five times. These cells were collected after incubation with PBS containing trypsin (0.1 mg/ml) and 2 mM EDTA for 15 min. The 1.8 x lo6cells/tube were cultured in a plastic tube containing 5 ml of RPMI-1640 with 10 per cent FCS in the presence or absence of agents. After the culture, cell smears were prepared as described above. Immunocytochemistry Smears were fixed in absolute ethanol. Cell blocks were fixed in acetone at - 20°C for more than 24 h, dehydrated in ethylene glycol monoethyl ethel (Katayama Chemical Corp., Osaka, Japan) at room temperature for 24 h, immersed in xylene for 2 x 30 min, and then embedded in paraffin.', The cell smears and deparaffinized sections were stained immunocytochemically for ACT, using rabbit antiACT antibody (Dako Corp., Santa Barbara, CA) at 1 : 10 000 dilution and a Vecstatin avidinbiotin-peroxidase kit (Vector Laboratories Inc., Burlingam, CA). Macrophages were identified by their ability to ingest Indian ink.I3 For determination of the cell populations, some smears and sections were stained with haematoxylin and eosin. Total cells, macrophages, and various cells were counted with smears in experiment I, 111, and IV, and with sections in experiment 11. There were no significant differences in the numbers or the percentages of cells among the various culture conditions in experiments I and 11. Macrophages occupied more than 98 per cent of cultured cells in experiments I11 and IV. Assay of alpha-1 antichymotrypsin in medium The medium was stored at - 80°C after centrifugation at 500g for 5 min. ACT was assayed at Mitubishi Yuca Bioclinical Laboratories, Osaka, Japan, by radioimmunoassay using sheep antihuman ACT and human ACT labelled with12'I by the chloramine T method. The separation of bound and free ['251] ACT was carried out with Immunobeads. RESULTS Experiment I: the efSect of P M A or LPS on the percentage of ACT-positive macrophages The percentages of alveolar macrophages with intracellular ACT in experiment I are shown in
32 1
ALPHA-I ANTICHYMOTRYPSIN IN ALVEOLAR MACROPHAGES
Fig. 1. The mean percentages in cultures with no agent and with PMA, dexamethasone, and LPS were 35,53,43, and 52 per cent, respectively, the increases induced by PMA and LPS being significant.
i
60
n
o\" W
Q)
L
40
Q
0
L
-I-
0
!i
r
T
PMA
DEX
*
LPS and LPSfDEX were 53, 36, 54, and 39 per cent, respectively. Although PMA, LPS, and DEX were added to the medium at half the concentrations used in experiment I, PMA and LPS both increased the percentage of ACT-positive macrophages significantly, and DEX completely suppressed these increases.
1
I
-r
6oI T
I
I
I
b
T I
Q)
.-.-w>
g
20
& 0 a 0
None
-
-
LPS
Fig. I-Effect of PMA, LPS, and dexamethasone o n the proportion of ACT-positive macrophages in experiment I. Alveolar macrophages obtained by bronchoalveolar lavage were cultured in suspension for 3 days in medium containing the indicated agents. The concentrations (in pg/ml) of PMA, DEX, and LPS were 0.2, 0.04, and 10, respectively. The number of each group was seven. Total number (mean _+ SE) of cells in cultures with no agent, and with PMA, DEX, and LPS: 2436*757, 1327k382, 2014k 1174, 1516k370; number of macrophages examined in each group: 615+ 147,482k 153, 582+280, 524+ 167; percentage (mean & SE) of macrophages in each group: 77 4, 77 & 6, 77 k 6, 68 & 9; percentage of neutrophils in each group: 1 k I , 0, 1 I , 3 i I ; percentage of lymphocytes in each group: 10&3, 13+4, IOk3, 17+7. PMA=phorbol myristate acetate; LPS= lipopolysaccharide; DEX = dexamethasone; ACT = alpha- I antichymotrypsin.* P c 0 . 0 5 vs. value of cultures with no agent (by Student's t-test)
*
Experiment 11: the eflect of dexamethasone on the percentage of ACT-positive macrophages The percentages of alveolar macrophages with intracellular ACT in experiment I1 are shown in Fig. 2 . The mean percentages ofmacrophages with intracellular ACT in cultures with PMA, PMA DEX,
+
OL -
~
-
~~
-
LPSGEX
Fig. 2-Effect of dexamethasone on the proportion of ACTpositive macrophages in experiment 11. Alveolar macrophages obtained by bronchoalveolar lavage were cultured in suspension for 3 days in medium containing the indicated agents. The concentrations (in ,ug/ml) of PMA, DEX, and LPS were 0.1, 0.02, and 5, respectively. The number of each group was seven. The total number of cells was not determined. Number (meankSE) of macrophages examined in cultures with PMA, PMA DEX, LPS, and L P S f D E X : 774k 160,780+ 147, 1096*310, 1528k 359; percentage (mean k SE) of macrophages in each group: 7 2 k 6 , 74+5, 70+7, 73+6; percentage of neutrophils in each group: 1 k I , I +0,3 2, 1 & I ; percentage of lymphocytes in each group: 9 k 3 , 9 k 3 , 1 3 k 5 , 10*4. dPp
165 319-323 (1991)
ALPHA-1 ANTICHYMOTRYPSIN IS INCREASED IN HUMAN ALVEOLAR MACROPHAGES BY PHORBOL MYRISTATE ACETATE OR LIPOPOLYSACCHARIDE A N D RELEASED FROM THESE ACTIVATED MACROPHAGES BY GLUCOCORTICOID TOMOFUMI NAGAREDA, MASASHI TAKEDA, KAYO KOJIMA, AKIRA TANAKA, NOBUYUKI TERADA, TOMOYOSHI YAMASAKI, TAEKO NAGAREDA, HIROSHI UENO A N D KIYOSHI KOTOH
Department of Pathology, Osaka University Medical School, Fukushima-ku, Osaka, 553; Department of Pathology. Osaka Medical College, Takatsuki. Osaka, 569, Japan Received 9 October 1990 Accepted 13 June 1991
SUMMARY Alveolar macrophages were obtained from 23 patients and the effects of phorbol myristate acetate (PMA),lipopolysaccharide (LPS), and dexamethasone (DEX) on the proportion of cells with intracellular alpha-1 antichymotrypsin (ACT), and concentrations of ACT in the culture medium were studied in vitro. The alveolar macrophages were obtained by bronchoalveolar lavage at autopsy or from resected lungs at operation and were cultured in suspension for 3 days in medium containing PMA, LPS, DEX, PMA+DEX, or LPS+DEX. Both PMA and LPS significantly increased the percentage of macrophages with intracellularACT. Dexamethasone did not increase the number of ACTpositive cells and significantly suppressed the increaseinduced by PMA or LPS, releasing ACT into the culture medium. The release of ACT from macrophages may contribute to the anti-inflammatoryeffects of corticoids. KEY WORDS-Human
alveolar macrophage, alpha-1 antichymotrypsin,phorbol myristate acetate, LPS glucocorticoid.
INTRODUCTION Alveolar macrophages play a major role in pulmonary defence. The actions of some macrophages require direct cell-to-cell interaction, whereas those of others are mediated through secretion of various substances such as prosta landin,’ complement component,2growth factors,K.~nt e rfe ron,~ and interleukin 1.5 Alveolar macrophages can also synthesize and secrete alpha-] antichymotrypsin (ACT),6 which is a glycoprotein that increases in amount in the acute phase of inflammati~n.’~~ Pulmonary inflammation is induced experimentally by phorbol myristate acetate (PMA) or lipopolysaccharide (LPS): PMA induces interstitial pneumonitis’ while LPS induces pneumonia.” Addressee for correspondence: Dr Tomofumi Nagareda, Oomino 91-36, Sakai, Osaka, 588, Japan.
0022-34 1 719 1 / I 203 19-05 $05.00 0 1991 by John Wiley & Sons, Ltd.
Glucocorticoids suppress inflammation of the lung.” In the present study, we investigated the in vitro effects of PMA, LPS, and glucocorticoid on the proportion of human alveolar macrophages with intracellular ACT and the concentration of ACT in the culture medium. Intracellular ACT was evaluated by immunocytochemical staining of ACT. MATERIALS AND METHODS
Collection of bronchoulveolurluvugejuid Human alveolar macrophages were obtained by bronchoalveolar lavage of 21 right lungs at autopsy and of a right and a left lobe removed surgically from two patients, both with squarnous cell carcinoma. These 23 patients ranged from 40 to 82 years old. The primary diseases of the autopsy cases were
320
T. NAGAREDA ET AL.
hepatocellular carcinoma with liver cirrhosis (7 cases), myocardial i n f a r ~ t i o ndiabetes ,~ mellitus (3), gastric cancer (2), renal failure (I), metastatic brain tumour from breast cancer (I), peritonitis (I), pancreatic cancer (I), laryngeal cancer (I), and cryptococcosis. (1) The pathological findings in the lungs were focal pneumonia in 10 cases, pulmonary oedema in 8, emphysema in 2, metastatic carcinoma in 2, and congestion in 1. Lavage was carried out ten times by instillation of sterile saline (5 ml) into bronchi S' or S3 in autopsy cases within 13 h after death and SIo in the operation cases, and gentle aspiration of fluid was carried out with a syringe. These bronchi and segments were all free from severe pulmonary lesions. Culture of macrophages in experiments I and I1 The bronchoalveolar lavage fluid was obtained from patients 1-7 in experiment I and from patients 8-14 in experiment IT. The lavage fluid was centrifuged at 500g for 5 min, and the cell pellet was washed twice with 5 ml of RPMI-1640 medium supplemented with 10 per cent fetal calf serum (FCS) and finally suspended in 4ml of RPMI-1640 medium with 10 per cent FCS. Samples of 1 ml of the cell suspension were introduced into individual plastic tubes with 4 ml of RPMI-I 640 medium containing 10 per cent FCS with or without PMA (Consolidated Midland Corp., Katonah, NY), LPS (Paesel GmbH, Germany), dexamethasone (DEX), PMA + DEX, or LPS DEX, and the macrophages was finally cultured in 5 ml of medium. The concentrations (in pg/ml) of PMA, LPS, and DEX were, respectively, 0.2, 10, and 0.04 in experiment I; and 0.1, 5 and 0.02 in experiment 11. The final concentrations of the agents in experiment I1 were half of those in experiment I. Macrophages were cultured in suspension for 3 days under 5 per cent CO, in air at 37°C. Then, 2Opl of Indian ink was introduced into each tube, and 15 min later the cells were collected by centrifugation at 500g for 5 min and the cell pellet was washed twice with phosphatebuffered saline (PBS, pH 7.2). In experiment I, cells were smeared on a slide glass and in experiment 11, a cell block was made by adding 40p1 of human serum, 2 W O pl of 0.025 mM calcium chloride, and 2 0 4 0 pl of 200 p/ml thrombin.
+
Culture of macrophages in experiments Illand IV The lavage fluid was obtained from patients 1518 in experiment I11 and from patients 19-23 in experiment IV. Cells in the lavage fluid were collected as described above. The cell suspension in
PBS was transferred into an uncoated plastic culture dish and incubated in 5 per cent CO, air at 37°C for 30 min. Then the medium was removed and macrophages attached to the dish well were washed with PBS five times. These cells were collected after incubation with PBS containing trypsin (0.1 mg/ml) and 2 mM EDTA for 15 min. The 1.8 x lo6cells/tube were cultured in a plastic tube containing 5 ml of RPMI-1640 with 10 per cent FCS in the presence or absence of agents. After the culture, cell smears were prepared as described above. Immunocytochemistry Smears were fixed in absolute ethanol. Cell blocks were fixed in acetone at - 20°C for more than 24 h, dehydrated in ethylene glycol monoethyl ethel (Katayama Chemical Corp., Osaka, Japan) at room temperature for 24 h, immersed in xylene for 2 x 30 min, and then embedded in paraffin.', The cell smears and deparaffinized sections were stained immunocytochemically for ACT, using rabbit antiACT antibody (Dako Corp., Santa Barbara, CA) at 1 : 10 000 dilution and a Vecstatin avidinbiotin-peroxidase kit (Vector Laboratories Inc., Burlingam, CA). Macrophages were identified by their ability to ingest Indian ink.I3 For determination of the cell populations, some smears and sections were stained with haematoxylin and eosin. Total cells, macrophages, and various cells were counted with smears in experiment I, 111, and IV, and with sections in experiment 11. There were no significant differences in the numbers or the percentages of cells among the various culture conditions in experiments I and 11. Macrophages occupied more than 98 per cent of cultured cells in experiments I11 and IV. Assay of alpha-1 antichymotrypsin in medium The medium was stored at - 80°C after centrifugation at 500g for 5 min. ACT was assayed at Mitubishi Yuca Bioclinical Laboratories, Osaka, Japan, by radioimmunoassay using sheep antihuman ACT and human ACT labelled with12'I by the chloramine T method. The separation of bound and free ['251] ACT was carried out with Immunobeads. RESULTS Experiment I: the efSect of P M A or LPS on the percentage of ACT-positive macrophages The percentages of alveolar macrophages with intracellular ACT in experiment I are shown in
32 1
ALPHA-I ANTICHYMOTRYPSIN IN ALVEOLAR MACROPHAGES
Fig. 1. The mean percentages in cultures with no agent and with PMA, dexamethasone, and LPS were 35,53,43, and 52 per cent, respectively, the increases induced by PMA and LPS being significant.
i
60
n
o\" W
Q)
L
40
Q
0
L
-I-
0
!i
r
T
PMA
DEX
*
LPS and LPSfDEX were 53, 36, 54, and 39 per cent, respectively. Although PMA, LPS, and DEX were added to the medium at half the concentrations used in experiment I, PMA and LPS both increased the percentage of ACT-positive macrophages significantly, and DEX completely suppressed these increases.
1
I
-r
6oI T
I
I
I
b
T I
Q)
.-.-w>
g
20
& 0 a 0
None
-
-
LPS
Fig. I-Effect of PMA, LPS, and dexamethasone o n the proportion of ACT-positive macrophages in experiment I. Alveolar macrophages obtained by bronchoalveolar lavage were cultured in suspension for 3 days in medium containing the indicated agents. The concentrations (in pg/ml) of PMA, DEX, and LPS were 0.2, 0.04, and 10, respectively. The number of each group was seven. Total number (mean _+ SE) of cells in cultures with no agent, and with PMA, DEX, and LPS: 2436*757, 1327k382, 2014k 1174, 1516k370; number of macrophages examined in each group: 615+ 147,482k 153, 582+280, 524+ 167; percentage (mean & SE) of macrophages in each group: 77 4, 77 & 6, 77 k 6, 68 & 9; percentage of neutrophils in each group: 1 k I , 0, 1 I , 3 i I ; percentage of lymphocytes in each group: 10&3, 13+4, IOk3, 17+7. PMA=phorbol myristate acetate; LPS= lipopolysaccharide; DEX = dexamethasone; ACT = alpha- I antichymotrypsin.* P c 0 . 0 5 vs. value of cultures with no agent (by Student's t-test)
*
Experiment 11: the eflect of dexamethasone on the percentage of ACT-positive macrophages The percentages of alveolar macrophages with intracellular ACT in experiment I1 are shown in Fig. 2 . The mean percentages ofmacrophages with intracellular ACT in cultures with PMA, PMA DEX,
+
OL -
~
-
~~
-
LPSGEX
Fig. 2-Effect of dexamethasone on the proportion of ACTpositive macrophages in experiment 11. Alveolar macrophages obtained by bronchoalveolar lavage were cultured in suspension for 3 days in medium containing the indicated agents. The concentrations (in ,ug/ml) of PMA, DEX, and LPS were 0.1, 0.02, and 5, respectively. The number of each group was seven. The total number of cells was not determined. Number (meankSE) of macrophages examined in cultures with PMA, PMA DEX, LPS, and L P S f D E X : 774k 160,780+ 147, 1096*310, 1528k 359; percentage (mean k SE) of macrophages in each group: 7 2 k 6 , 74+5, 70+7, 73+6; percentage of neutrophils in each group: 1 k I , I +0,3 2, 1 & I ; percentage of lymphocytes in each group: 9 k 3 , 9 k 3 , 1 3 k 5 , 10*4. dPp
