Int. Archs Allergy appl. Immun. 49: 585-596 (1975)
Skin Reaction, Inhibition of Macrophage Migration, and Lymphocyte Transformation with Tuberculin Active Peptide (TAP) and Arabinogalactan Obtained from Tubercle Bacilli T. N iinaka, S. K ishimoto , T. A oki, H. I kegami, F. Ito and Y. Y amamura Third Department of Internal Medicine, Osaka University School of Medicine, Osaka
Abstract. Arabinogalactan purified from heat-killed tubercle bacilli failed to elicit a delayed type of skin reaction and had no ability to induce in vitro lympho cyte blast formation in sensitized guinea pigs. It was, however, inhibitory to mi gration of bronchoalveolar washing cells by the indirect test, but not by the direct test, and capable of eliciting an immediate type of skin reaction and anaphylaxis when injected into sensitized guinea pigs. Tuberculin active peptide (TAP) was active in all of in vitro and in vivo cellmediated immune responses, but not in immediate responses.
In previous papers [2, 3, 4] we reported the chemical and immunol ogical properties of polysaccharides, including glucan, mannan, arabinomannan and arabinogalactan (Ara-Gal) purified from Aoyama B strain of human tubercle bacilli. Each of these purified polysaccharides was completely free from protein and as much as 100//g of these polysac charides were incapable of eliciting a delayed type of skin reaction, whereas Ara-Gal and arabinomannan could induce anaphylaxis and an Arthus-type skin reaction in guinea pigs sensitized with heat-killed tub ercle bacilli. On the other hand, tuberculin active peptide (TAP) [15, 18] extract ed from the same strain of tubercle bacilli is known to elicit a typical delayed type, but no immediate type of skin reaction, similar to that in duced by PPDs. In contrast, some investigators claim [5, 6, 9, 10] that polysaccharides, though not completely free from protein, obtained from tubercle bacilli are also capable of eliciting a delayed type of skin reac-
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Received: January 2,1975.
586
N iinaka/K ishimoto /A oki/I kegami/I to/Y amamura
tion in sensitized guinea pigs. Furthermore, evidence was presented by C haparas’s team [5, 6, 10] that polysaccharides obtained from an un heated culture filtrate of tubercle bacilli are able to inhibit the migration of macrophages from guinea pigs immunized with tubercle bacilli. In this context, this paper will present some evidence that Ara-Gal purified from tubercle bacilli is not capable of eliciting a delayed type of skin reaction, inhibiting migration of alveolar macrophages by the direct test, and of inducing lymphocyte blast formation in guinea pigs immu nized with tubercle bacilli; however, inhibition of macrophage migration is observed by the indirect test. Materials and Methods
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Animals and sensitization. Male guinea pigs of Hartley strain, weighing 350+50 g were obtained from local suppliers. Animals were sensitized by inocu lating 2 mg of heat-killed tubercle bacilli suspended in 1 ml of liquid paraffin arlacel (85:15) into the footpads and boosted similarly 3 weeks later. These animals were used for experiments 3 or 4 weeks after booster immunization. Antigens. TAP and Ara-Gal were extracted from Aoyama B strain of human tubercle bacilli and purified as described previously [2-4, 15, 18]. Purified AraGal was positive in the phenol sulfuric acid reaction but negative in the ninhydrin and Lowry reaction. An elementary analysis showed this polysaccharide to be composed of C:41.36 H:6.61 and only traces of nitrogen. Arabinose and galactose were shown to be present in a 14.8:1.0 molar ratio. Lymphokine preparation [1, 17]. Cervical, axillary and inguinal lymph nodes were removed aseptically from sensitized guinea pigs, teased gently and filtered through a stainless wire mesh into Eagle’s minimum essential medium (EMEM). After washing, the cells were resuspended in EMEM at a concentration of 1-3 X107 cells/ml, which was confirmed to contain 1.3-2 X106 viable cells. 5 ml of this cell suspension were cultured in the presence of either TAP or Ara-Gal at a concentration of 15 «g/ml at 37 °C for 48 h in an incubator gassed with 5°/o CO, in air. After incubation, supernatants were obtained by centrifuging the cell cul ture medium and the residues after dialysis were lyophilized. This is hereinafter referred to as the ‘crude lymphokines’. Preparation of partially purified migration inhibitory factor (MIF). The crude lymphokines were rcdissolved in distilled water, clarified by centrifugation, and fractionated by gel filtration through a column of Sephadex G-100. The fractions of peak IV (fig. 4) were pooled, centrifuged and lyophilized. These partially puri fied materials are hereinafter referred to as ‘LK ( )’, each of which denotes the kind of the antigens used for the production of lymphokines in parenthesis. The fractionation procedure is summarized in figure 3. Macrophage migration inhibition test. Both direct and indirect tests were car ried out with the use of the bronchoalveolar washing cells derived from guinea pigs.
Skin Reaction, Inhibition of Macrophage Migration
587
Direct test. It was essentially similar to that of G eorge and V aughan [8]. In brief, the bronchoalveolar washing cells from lungs of sensitized guinea pigs were collected into a heparinized flask according to the technique of M yrvik et al. [14]. These cells were adjusted to a concentration of 107 cells/ml in EMEM containing 20% fetal calf serum. Hematocrit capillaries were filled with the cell suspension, plugged with clay at one end, and centrifuged for 8 min at 800 rpm. Then they were cut at the cell-medium interface and the parts containing packed cells were held in place on the bottom coverslip of the Mackaness-type chamber, two per chamber with a spot of silicone grease. After the top coverslip was sealed with paraffin, the chamber was filled with medium alone or medium containing an antigen and incubated at 37 °C for 48 h. TAP or Ara-Gal was added to give a final concentration of 15 «g/ml in culture medium. 24 and 48 h after incubation, the areas of migration were photographed at 10-fold magnification, cut out and weighed. The following formula was used to express the migration index (MI): weight of average area of migration with antigen MI = -----------------------------------------------------------------X 100. weight of average area of migration without antigen Indirect test. Indicator cells used were bronchoalveolar washing cells from nor mal guinea pigs. The procedure was similar to that used for the direct test except that crude or partially purified lymphokines were added to the culture medium at a final concentration of 1 mg/ml. The reconstitution was carried out according to D umonde et al. [7]. These MI tests were carried out in duplicate three times at least and no remarkable discrepancy was found to occur among them. Only typi cal data were shown in this paper. In vitro test of lymphocyte blast formation [12]. Lymph nodes from guinea pigs sensitized with tubercle bacilli were removed aseptically and the cell concen tration was adjusted to 2.5X106/ml in RPMI 1640 medium containing 10% fetal calf serum. The degree of blast formation was determined by uptake of tritiated thymidine into DNA of lymph node cells. The test tubes which received 2 ml of cell suspension with or without 50,«g of either TAP or Ara-Gal, were kept at 37 °C for 72 h in an incubator gassed with 5% COz in air at 100% humidity. 24 h before harvesting the cells, 1 pCi of tritiated thymidine was added to all tubes. For radioassaying tritiated thymidine incorporated into DNA, cells were washed thrice with cold 5% trichloroacetic acid and then once with ethanol. The radioactivity was counted in a liquid scintillation counter. The data were expressed as the mean of duplicate samples. An index of the response to antigens was calculated by comparison with that of unstimulated cells.
Results
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Anaphylaxis and Skin Reaction in Sensitized Guinea Pigs by TAP or Ara-Gal As shown in table I, Ara-Gal possesses a strong activity to induce anaphylactic reaction in actively sensitized guinea pigs. The sensitized
588
N iinaka/K ishimoto /A oki/I kegami/I to/Y amamura
Table I. Anaphylactic shock activity of purified polysaccharide (Ara-Gal) Active anaphylaxis antigen dose mg
symptoms
1.0 1.0 0.5 0.5 0.1 0.1 0.05
++ ++ ++ ++ ++ ++ ++
Saline 1.0
-
+ + + + + +
Passive anaphylaxis antiserum, ml
3.0 3.0 3.0 1.0 1.0 1.0 0.5 0.5 0.5
symptoms
+ + + + + +
++ ++ ++ + + +
_L
+ -f.
Normal serum1 5.0 5.0 + + + = Died within 5 min; + + = guinea pigs survived with serious symptoms, such as cramp, dyspnea, and cyanosis; + = mild symptoms such as mild cramp, dyspnea, tachypnea, and erectio pili; — = no anaphylactic sign. 1 Antigen, 2 mg.
guinea pigs presented serious anaphylactic signs and died within 5 min after an intravenous injection of 100 pg of Ara-Gal. Figure 1 shows the time course of skin reactions elicited by Ara-Gal or TAP in sensitized guinea pigs. Erythema and wheal began to appear at the injected site of skin 1 h after injection of Ara-Gal, reached the peak around 5 h and disappeared completely after 48 h. On the other hand, TAP induced typical delayed skin reactions, which attained the maximum around 24 h after injection. These findings indicate that the purified Ara-Gal is capa ble of eliciting an immediate type reaction but no delayed type reaction in sensitized animals. By contrast, TAP can induce only a delayed type reaction.
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Direct Test for Macrophage Migration Inhibition with TAP or AraGal First, to ascertain whether TAP or Ara-Gal may exert any effect on the migration of normal bronchoalveolar washing cells, the direct test was carried out. As shown in table II, TAP or Ara-Gal was not or little
Skin Reaction, Inhibition of Macrophage Migration
589
Fig. 1. Skin reaction after intradermal injection of purified polysaccharide (Ara-Gal, • ), or TAP (A) in sensitized guinea pigs. Table II. Effect of TAP, PPDs and purified polysaccharide (Ara-Gal) on the capillary migration of normal alveolar macrophages (48 h) Test
MI
No antigen TAP PPDs Ara-Gal
100 72 72 79
Table III. Ml of alveolar macrophages from tuberculin-sensitive guinea pig by TAP, PPDs and purified polysaccharide (Ara-Gal ; 48 h) Test
MI
No antigen TAP PPDs Ara-Gal
100 58 65 83
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inhibitory to the migration of normal bronchoalveolar washing cells. Thus, we attempted to clarify whether TAP or Ara-Gal may inhibit the migration of cells from sensitized animals. As shown in table III and fig ure 2, the migration of cells was strikingly inhibited in the presence of TAP, but not in the presence of Ara-Gal, when bronchoalveolar wash ing cells from a sensitized guinea pig was used. TAP was strongly inhibi-
590
N
i i n a k a / K i s i i i m o t o /A o k i /I kf .g a m i /It o / Y a m a m u r a
No antigen
TAP
lO/ig/ml
Fig. 2. Effect of purified polysaccharide (Ara-Gal) on the capillary migration of alveolar macrophages from sensitized guinea pig (48 h).
Indirect Test for Macrophage Migration Inhibition with Crude or Partially Purified MIF Culture supernatant of sensitized lymph node cells incubated without TAP or Ara-Gal was not inhibitory to cell migration. As another con trol, immediately after the culture of sensitized lymph node cells without TAP or Ara-Gal, the medium was reconstituted with each antigen. This control also did not show any inhibition on macrophage migration. In contrast, crude lymphokines prepared from culture medium of sensitized lymph node cells incubated with TAP induced moderate inhibition of migration, whereas those prepared from medium of lymph node cells in cubated with Ara-Gal did not (shown in table IV). As the next step, partial purification of MIF from culture supernatants of sensitized lymph node cells incubated with TAP was attempted, as shown in fig ure 3 and 4. Five fractions of crude lymphokines were obtained by gel filtration through a column of Sephadcx G-I00. Among them fraction
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tory at a concentration of 10 /
Skin Reaction, Inhibition of Macrophage Migration, and Lymphocyte Transformation with Tuberculin Active Peptide (TAP) and Arabinogalactan Obtained from Tubercle Bacilli T. N iinaka, S. K ishimoto , T. A oki, H. I kegami, F. Ito and Y. Y amamura Third Department of Internal Medicine, Osaka University School of Medicine, Osaka
Abstract. Arabinogalactan purified from heat-killed tubercle bacilli failed to elicit a delayed type of skin reaction and had no ability to induce in vitro lympho cyte blast formation in sensitized guinea pigs. It was, however, inhibitory to mi gration of bronchoalveolar washing cells by the indirect test, but not by the direct test, and capable of eliciting an immediate type of skin reaction and anaphylaxis when injected into sensitized guinea pigs. Tuberculin active peptide (TAP) was active in all of in vitro and in vivo cellmediated immune responses, but not in immediate responses.
In previous papers [2, 3, 4] we reported the chemical and immunol ogical properties of polysaccharides, including glucan, mannan, arabinomannan and arabinogalactan (Ara-Gal) purified from Aoyama B strain of human tubercle bacilli. Each of these purified polysaccharides was completely free from protein and as much as 100//g of these polysac charides were incapable of eliciting a delayed type of skin reaction, whereas Ara-Gal and arabinomannan could induce anaphylaxis and an Arthus-type skin reaction in guinea pigs sensitized with heat-killed tub ercle bacilli. On the other hand, tuberculin active peptide (TAP) [15, 18] extract ed from the same strain of tubercle bacilli is known to elicit a typical delayed type, but no immediate type of skin reaction, similar to that in duced by PPDs. In contrast, some investigators claim [5, 6, 9, 10] that polysaccharides, though not completely free from protein, obtained from tubercle bacilli are also capable of eliciting a delayed type of skin reac-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
Received: January 2,1975.
586
N iinaka/K ishimoto /A oki/I kegami/I to/Y amamura
tion in sensitized guinea pigs. Furthermore, evidence was presented by C haparas’s team [5, 6, 10] that polysaccharides obtained from an un heated culture filtrate of tubercle bacilli are able to inhibit the migration of macrophages from guinea pigs immunized with tubercle bacilli. In this context, this paper will present some evidence that Ara-Gal purified from tubercle bacilli is not capable of eliciting a delayed type of skin reaction, inhibiting migration of alveolar macrophages by the direct test, and of inducing lymphocyte blast formation in guinea pigs immu nized with tubercle bacilli; however, inhibition of macrophage migration is observed by the indirect test. Materials and Methods
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
Animals and sensitization. Male guinea pigs of Hartley strain, weighing 350+50 g were obtained from local suppliers. Animals were sensitized by inocu lating 2 mg of heat-killed tubercle bacilli suspended in 1 ml of liquid paraffin arlacel (85:15) into the footpads and boosted similarly 3 weeks later. These animals were used for experiments 3 or 4 weeks after booster immunization. Antigens. TAP and Ara-Gal were extracted from Aoyama B strain of human tubercle bacilli and purified as described previously [2-4, 15, 18]. Purified AraGal was positive in the phenol sulfuric acid reaction but negative in the ninhydrin and Lowry reaction. An elementary analysis showed this polysaccharide to be composed of C:41.36 H:6.61 and only traces of nitrogen. Arabinose and galactose were shown to be present in a 14.8:1.0 molar ratio. Lymphokine preparation [1, 17]. Cervical, axillary and inguinal lymph nodes were removed aseptically from sensitized guinea pigs, teased gently and filtered through a stainless wire mesh into Eagle’s minimum essential medium (EMEM). After washing, the cells were resuspended in EMEM at a concentration of 1-3 X107 cells/ml, which was confirmed to contain 1.3-2 X106 viable cells. 5 ml of this cell suspension were cultured in the presence of either TAP or Ara-Gal at a concentration of 15 «g/ml at 37 °C for 48 h in an incubator gassed with 5°/o CO, in air. After incubation, supernatants were obtained by centrifuging the cell cul ture medium and the residues after dialysis were lyophilized. This is hereinafter referred to as the ‘crude lymphokines’. Preparation of partially purified migration inhibitory factor (MIF). The crude lymphokines were rcdissolved in distilled water, clarified by centrifugation, and fractionated by gel filtration through a column of Sephadex G-100. The fractions of peak IV (fig. 4) were pooled, centrifuged and lyophilized. These partially puri fied materials are hereinafter referred to as ‘LK ( )’, each of which denotes the kind of the antigens used for the production of lymphokines in parenthesis. The fractionation procedure is summarized in figure 3. Macrophage migration inhibition test. Both direct and indirect tests were car ried out with the use of the bronchoalveolar washing cells derived from guinea pigs.
Skin Reaction, Inhibition of Macrophage Migration
587
Direct test. It was essentially similar to that of G eorge and V aughan [8]. In brief, the bronchoalveolar washing cells from lungs of sensitized guinea pigs were collected into a heparinized flask according to the technique of M yrvik et al. [14]. These cells were adjusted to a concentration of 107 cells/ml in EMEM containing 20% fetal calf serum. Hematocrit capillaries were filled with the cell suspension, plugged with clay at one end, and centrifuged for 8 min at 800 rpm. Then they were cut at the cell-medium interface and the parts containing packed cells were held in place on the bottom coverslip of the Mackaness-type chamber, two per chamber with a spot of silicone grease. After the top coverslip was sealed with paraffin, the chamber was filled with medium alone or medium containing an antigen and incubated at 37 °C for 48 h. TAP or Ara-Gal was added to give a final concentration of 15 «g/ml in culture medium. 24 and 48 h after incubation, the areas of migration were photographed at 10-fold magnification, cut out and weighed. The following formula was used to express the migration index (MI): weight of average area of migration with antigen MI = -----------------------------------------------------------------X 100. weight of average area of migration without antigen Indirect test. Indicator cells used were bronchoalveolar washing cells from nor mal guinea pigs. The procedure was similar to that used for the direct test except that crude or partially purified lymphokines were added to the culture medium at a final concentration of 1 mg/ml. The reconstitution was carried out according to D umonde et al. [7]. These MI tests were carried out in duplicate three times at least and no remarkable discrepancy was found to occur among them. Only typi cal data were shown in this paper. In vitro test of lymphocyte blast formation [12]. Lymph nodes from guinea pigs sensitized with tubercle bacilli were removed aseptically and the cell concen tration was adjusted to 2.5X106/ml in RPMI 1640 medium containing 10% fetal calf serum. The degree of blast formation was determined by uptake of tritiated thymidine into DNA of lymph node cells. The test tubes which received 2 ml of cell suspension with or without 50,«g of either TAP or Ara-Gal, were kept at 37 °C for 72 h in an incubator gassed with 5% COz in air at 100% humidity. 24 h before harvesting the cells, 1 pCi of tritiated thymidine was added to all tubes. For radioassaying tritiated thymidine incorporated into DNA, cells were washed thrice with cold 5% trichloroacetic acid and then once with ethanol. The radioactivity was counted in a liquid scintillation counter. The data were expressed as the mean of duplicate samples. An index of the response to antigens was calculated by comparison with that of unstimulated cells.
Results
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
Anaphylaxis and Skin Reaction in Sensitized Guinea Pigs by TAP or Ara-Gal As shown in table I, Ara-Gal possesses a strong activity to induce anaphylactic reaction in actively sensitized guinea pigs. The sensitized
588
N iinaka/K ishimoto /A oki/I kegami/I to/Y amamura
Table I. Anaphylactic shock activity of purified polysaccharide (Ara-Gal) Active anaphylaxis antigen dose mg
symptoms
1.0 1.0 0.5 0.5 0.1 0.1 0.05
++ ++ ++ ++ ++ ++ ++
Saline 1.0
-
+ + + + + +
Passive anaphylaxis antiserum, ml
3.0 3.0 3.0 1.0 1.0 1.0 0.5 0.5 0.5
symptoms
+ + + + + +
++ ++ ++ + + +
_L
+ -f.
Normal serum1 5.0 5.0 + + + = Died within 5 min; + + = guinea pigs survived with serious symptoms, such as cramp, dyspnea, and cyanosis; + = mild symptoms such as mild cramp, dyspnea, tachypnea, and erectio pili; — = no anaphylactic sign. 1 Antigen, 2 mg.
guinea pigs presented serious anaphylactic signs and died within 5 min after an intravenous injection of 100 pg of Ara-Gal. Figure 1 shows the time course of skin reactions elicited by Ara-Gal or TAP in sensitized guinea pigs. Erythema and wheal began to appear at the injected site of skin 1 h after injection of Ara-Gal, reached the peak around 5 h and disappeared completely after 48 h. On the other hand, TAP induced typical delayed skin reactions, which attained the maximum around 24 h after injection. These findings indicate that the purified Ara-Gal is capa ble of eliciting an immediate type reaction but no delayed type reaction in sensitized animals. By contrast, TAP can induce only a delayed type reaction.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
Direct Test for Macrophage Migration Inhibition with TAP or AraGal First, to ascertain whether TAP or Ara-Gal may exert any effect on the migration of normal bronchoalveolar washing cells, the direct test was carried out. As shown in table II, TAP or Ara-Gal was not or little
Skin Reaction, Inhibition of Macrophage Migration
589
Fig. 1. Skin reaction after intradermal injection of purified polysaccharide (Ara-Gal, • ), or TAP (A) in sensitized guinea pigs. Table II. Effect of TAP, PPDs and purified polysaccharide (Ara-Gal) on the capillary migration of normal alveolar macrophages (48 h) Test
MI
No antigen TAP PPDs Ara-Gal
100 72 72 79
Table III. Ml of alveolar macrophages from tuberculin-sensitive guinea pig by TAP, PPDs and purified polysaccharide (Ara-Gal ; 48 h) Test
MI
No antigen TAP PPDs Ara-Gal
100 58 65 83
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
inhibitory to the migration of normal bronchoalveolar washing cells. Thus, we attempted to clarify whether TAP or Ara-Gal may inhibit the migration of cells from sensitized animals. As shown in table III and fig ure 2, the migration of cells was strikingly inhibited in the presence of TAP, but not in the presence of Ara-Gal, when bronchoalveolar wash ing cells from a sensitized guinea pig was used. TAP was strongly inhibi-
590
N
i i n a k a / K i s i i i m o t o /A o k i /I kf .g a m i /It o / Y a m a m u r a
No antigen
TAP
lO/ig/ml
Fig. 2. Effect of purified polysaccharide (Ara-Gal) on the capillary migration of alveolar macrophages from sensitized guinea pig (48 h).
Indirect Test for Macrophage Migration Inhibition with Crude or Partially Purified MIF Culture supernatant of sensitized lymph node cells incubated without TAP or Ara-Gal was not inhibitory to cell migration. As another con trol, immediately after the culture of sensitized lymph node cells without TAP or Ara-Gal, the medium was reconstituted with each antigen. This control also did not show any inhibition on macrophage migration. In contrast, crude lymphokines prepared from culture medium of sensitized lymph node cells incubated with TAP induced moderate inhibition of migration, whereas those prepared from medium of lymph node cells in cubated with Ara-Gal did not (shown in table IV). As the next step, partial purification of MIF from culture supernatants of sensitized lymph node cells incubated with TAP was attempted, as shown in fig ure 3 and 4. Five fractions of crude lymphokines were obtained by gel filtration through a column of Sephadcx G-I00. Among them fraction
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 8:49:08 AM
tory at a concentration of 10 /
