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activation protein-1 (NAP-1) causes human basophil histamine release
Immunology Letters, 22 (1989) 151-154 Elsevier IMLET 01265
Neutrophil attractant/activation protein-1 (NAP-l) causes human basophil histamine release M a r t h a V. W h i t e 1, Teizo Y o s h i m u r a 3, William H o o k 2, M i c h a e l A. Kaliner I a n d E d w a r d J. Leonard 3 1National Institute of Allergy and Infectious Diseases, Building 10, Room 11C205, Bethesda, Maryland 20892, U.S.A.; 2National Institute of Dental Research, Building 10, Room 1A26, Bethesda, Maryland 20892, U.S.A.; 3National Cancer Institute-FCRF, Building 560, Room 12-71, Frederick, Maryland 21701, U.S.A. (Received 9 January 1989; revision received 24 April 1989; accepted 9 May 1989)
1. Summary
Basophils from five of six human donors released histamine in response to neutrophil attractant/activation protein-1 (NAP-l). Histamine release by this protein was concentration-dependent over the range of 3 x 10-7 M to 4 x 10-6 M. At 4 x 10-6 M, the mean agonist-induced release was 16_+3% (SEM) of total basophil histamine. For the same basophil preparations, release by anti-IgE was 35+6°7o. The chemotactic protein did not cause release of histamine from basophils at 0°C or in the presence of 10 mM EDTA. The time-course of histamine release was rapid; release was 43% o f maximal after 30 s and maximal after 1 min of incubation. Thus, in addition to its previously characterized neutrophil chemotactic and activating properties, this protein activates human basophils. 2. Introduction
The isolation and purification to homogeneity of
a human monocyte derived neutrophil attractant/activation protein (NAP-l) has recently been described [1-4]. Pure NAP-I can now be obtained from culture fluid of LPS-stimulated mononuclear cells in a 2-step procedure by affinity chromatography on a column of mouse monoclonal anti-NAP- 1, followed by C M - H P L C [5]. The cDNA for NAP-1 contains a coding sequence of 99 residues [6], the last 72 of which correspond to NAP- 1. Two more NAP-1 proteins, with 5 and 7 additional amino acids at the N-terminus, have been reported [5]. NAP-1 is not only a neutrophil chemoattractant, but also a secretory and metabolic stimulus, as shown by lysosomal enzyme release, superoxide and hydrogen peroxide production, and a rise in intracellular calcium ion [3, 7, 8]. Although NAP-1 has no chemotactic activity for monocytes or eosinophils, it attracts a subpopulation of human basophils (E. J. L., unpublished). This evidence for NAP-l-basophil interaction suggested that NAP-1 might induce basophil histamine release, especially in view of the fact that another protein, C5a, is both a chemoattractant [9] and a secretagogue [10] for human basophils.
Key words: Neutrophil attractant/activation protein-I; Histamine release; Basophils; Monocyte; Neutrophil; Chemotaxis
3. Materials and methods
Abbreviations." HBSS, Hanks balanced salt solution; HRA-N, neutrophil-derived histamine-releasing activity; NAP-l, neutrophil attractant activation protein; PMN, polymorphonuclear leukocyte.
3.1. NAP-1 production and isolation
Correspondence to." Martha V. White, National Institutes of Health, Building 10, Room 11C207, Bethesda, Maryland 20892, U.S.A.
NAP-1 was produced by LPS-stimulated human blood mononuclear cells and purified to homogeneity by'ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography [2]. The pure protein was stored in
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
151
35% acetonitrile at -80 °C. Prior to use, an aliquot was lyophilized and reconstituted in HBSS at a concentration of 6 x 10-6 M. Recombinant N A P - l , expressed in Escherichia coli, was supplied at a concentration of 1 m g / m l in 0.15 M NaCI by Dr. Kouji Matsushima, National Cancer Institute, Frederick, Maryland. 3.2. Preparation of human leukocytes Unfractionated leukocytes were obtained from venous blood of healthy volunteers by dextran sedimentation as previously described [11]. Blood was anticoagulated with EDTA and mixed with dextran 75 (Abbott Laboratories, North Chicago, IL). After an erythrocyte sedimentation period of 90 min, supernatant leukocytes were washed extensively in HBSS without calcium or magnesium. Just prior to histamine release experiments, cells were diluted in HBSS containing 3 m M Ca ÷ ÷ and 2 m M Mg ÷ ÷. In one experiment, basophils were separated from neutrophils by centrifugation of blood on density 1.077 Ficoll/Hypaque [12]. 3.3. Histamine release experiments To 12 x 75 polypropylene culture tubes were added 10/A aliquots of leukocyte suspension (20-40 ng total histamine) and 60/A of HBSS containing 3 mM Ca ++ (1.5 m M free Ca ++ by Ca ++ electrode measurement) and 2 m M Mg ÷ ÷ with or without secretagogue. After incubation at 37 °C for 45 min, the reaction was terminated by addition of 600 fzl HBSS without Ca ÷ ÷ or Mg ÷÷ . Cells were separated from incubation fluid by centrifugation at 4°C. Histamine content of cells and supernatants was determined by an automated fluorometric assay which is sensitive to 0.2 n g / m l [13]. Results are expressed as % histamine release corrected for spontaneous release (less than 5%) in the presence of buffer alone.
4. Results and discussion
NAP-1 caused histamine release by washed leukocyte preparations from 5 of 6 human donors tested (Fig. 1). At the highest concentration, net release was 16+_3% (SEM for 4 preparations). The 5 N A P - l responsive leukocyte preparations also released histamine when incubated with anti-IgE (0.1-1 #g/ml); net maximal release was 35_+6%. Anti-IgE induced 41% release from leukocytes of the single non-atopic donor that failed to release histamine in response to NAP-1. There was no relationship between the atopic status of the donors and the sensitivity of their basophils to N A P - l - i n d u c e d histamine release. Nor was there a correlation either by regression analysis or S p e a r m a n ' s rank correlation between the magnitudes of maximal response to anti-IgE and NAP-1. This result is comparable to that of Siraganian and H o o k [14], who studied histamine release by C5a, fMet-Phe-Met and anti-IgE. Magnitude of release was different for each donor and agonist, and magnitude of histamine release by one agonist did not correlate with that induced by any other agonist. The concentration range over which NAP-1 caused release of histamine was two orders of magnitude higher than the optimal N A P - 1 concentration (10 -8 M) required for basophil chemotaxis (Leonard et al., unpublished). If we as20 ¸
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3.4. Allergic status of basophil donors Donors were defined as allergic if they had seasonal asthma or rhinitis with positive prick skin tests to aeroallergens corresponding to their symptoms, or if they had anaphylactic reactions to foods to which they were skin test-positive.
152
1
2
3
-6 NAP-1 CONCENTRATION X 10
4-
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Fig. 1. Histamine release in response to NAP- l by washed leukocytes from 2 atopic and 3 non-atopic donors. Cells were incubated 45 rain at 37 °C with various concentrations of N A P - 1 or buffer. Nine experiments were performed. Data points are means +_ SEM for 4 to 9 duplicate measurements at the indicated concentrations. The ordinate represents histamine release in the presence of agonist minus spontaneous release, as a percentage of total leukocyte histamine.
sume a minimum occupancy o f 5% of basophil NAP-1 receptors at an agonist concentration of 10-8 M, almost all receptors would be occupied in the 10-6 M concentration range that induced histamine release [15]. At least a 10-fold higher concentration of agonist for secretion than for chemotaxis was also required for neutrophil responses to fMetLeu-Phe [16]. The possibility that histamine release was caused by trace amounts of other cytokines in purified NAP-1 was addressed by assessing histamine release by 4 donors' basophils in response to recombinant NAP-1 (Fig. 2). All 4 donors' basophils responded to recombinant N A P - l , and the magnitude of histamine release was comparable to that induced by purified NAP-1. The data suggest that histamine release was caused by NAP-1 and not by contaminating cytokines. The time-course of NAP-l-induced basophil histamine release is illustrated in Fig. 3. Histamine release was 43% complete after 30 s and complete after 1 min of incubation. C5a and fMet-Phe-Met cause histamine release with an equally rapid time course [14]. This is in contrast to the time-course of basophil histamine release induced by anti-IgE, which is not complete until 20 min [17]. Several histamine releasing factors, including macrophage-
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Fig. 2. Histamine release in response to recombinant NAP-1 by washed leukocytes from 2 atopic and 2 non-atopic donors (3 of the donors were also used in Fig. 1). Cells were incubated 45 min at 37 °C with various concentrations of recombinant NAP-1 or buffer. Data points are means _+ SEM for 4 duplicate measurements at the indicated concentrations. The ordinate represents histamine release in the presence of agonist minus spontaneous release, as a percentage of total leukocyte histamine.
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Fig. 3. Time course of NAP-l-induced human basophil histamine release. Leukocytes were mixed with 4× 10-6 M NAP-1 at 37 °C. At different intervals, the reaction was stopped by the addition of ice cold 0.01 M EDTA. Tubes were centrifuged and net histamine release was determined. The data are expressed as mean _+ SEM for 4 experiments. Net maximal histamine release ranged from 14 to 26°7o.
derived histamine releasing factor, are thought to activate human basophils by interacting with IgE [18-20]. The time-course data suggest that NAP-1 does not work through this mechanism. Neutrophils spontaneously release a histamine releasing activity, termed HRA-N, which causes rapid release of basophil histamine [11, 12]. Since NAP1 is a PMN secretagogue, it was of interest to determine if NAP-l-induced histamine release was dependent upon or enhanced by the presence of PMN. Venous blood was processed as described in Section 3 to provide two leukocyte preparations, one of which was depleted of PMN. In experiments with leukocytes from 3 donors, 3 × 10 -6 M NAP-1 caused equivalent amounts of histamine release from both preparations of leukocytes (data not shown). Thus NAP-l-induced basophil histamine release is neither dependent upon nor enhanced by PMN, suggesting that NAP-1 acts directly to induce basophil activation rather than through HRA-N. Table 1 shows the temperature and divalent cation dependence of NAP-l-induced histamine release. Release was inhibited 750/0 in the presence of 0.01 M EDTA (4.5% compared to 16% net histamine release without EDTA, p < 0.025), indicating that N A P - I induced release rbquires divalent cations. No release occurred from basophils incubated with NAP-1 at 4 °C, whereas leukocytes from the same donors in153
TABLE 1
References
Temperature and divalent cation dependence of NAP-1-induced basophil histamine release.
[1] Yoshimura, T., Matsushima, K., Oppenheim, J. J. and Leonard, E. J. (1987) J. Immunol. 139, 788-793. [2] Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Appella E., Oppenheim, J. J. and Leonard, E. J. (1987) Proc. Natl. Acad. Sci. USA 84, 9233-9237. [3] Schroeder, J. M., Mrowietz, U., Morita, E. and Christophers, E. (1987) J. lmmunol. 139, 3474-3484. [4] Walz, A., Peveri, P., Aschauer, H. and Baggiolini, M. (1987) Biochem. Biophys. Res. Comm. 149, 755-761. [5] Yoshimura, T., Robinson, E. A., Appella, E., Matst/shima, K., Showalter, S. D., Skeel, A. and Leonard, E. J. (1988) Mol. lmmunol. In press. [6] Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, S. F., Leonard, E. J. and Oppenheim, J. J. (1988) J. Exp. Med. 167, 1883-1893. [7] Peveri, P., Walz, A., Dewald, B. and Baggiolini, M. (1988) J. Exp. Med. 167, 1547-1559. [8] Thelen, M., Peveri, P., Karnen, P., von Tscharner, V., Waltz, A. and Baggiolini, M. (1988) FASEB J. 2, 2702-2706. [9] Lett-Brown, M., Boetcher, D. A. and Leonard, E. J. (1976) J. lmmunol. 117, 246-252. [10] Hook, W., Siraganian, R. P. and Wahl, S. (1975) J. immunol. 114, 1185-1190. [11] White, M. V., Kaplan, A. P., Haak-Frendscho, M. and Kaliner, M. (1988) J. lmmunol. 141, 3575-3583. [12] White, M. V. and Kaliner, M. A. (1987) J. Immunol. 139, 1624-1630. [13] Siraganian, R. P. (1974) Anal. Biochem. 57, 383-394. [14] Siraganian, R. P. and Hook, W. A. (1977) J. lmmunol. 119, 2078-2083. [15] Waud, D. R. (1968) Pharmacol. Rev. 20, 49-88. [16] Snyderman, R. (1984) Federation Proc. 43, 2743-2748. [17] Gillespie, E. and Lichtenstein, L. M. (1972) J. Clin. Invest. 51, 2941-2947. [18] Liu, M. C., Proud, D., Lichtenstein, L. M., MacGlashlan Jr., D. W., Schleimer, R. P., Adkinson Jr., N. F., Kagey-Sobotka, A., Schulman, E. S. and Plaut, M. (1986) J. lmmunol. 136, 2588-2595. [19] Theuson, D. O., Speck, L. S., Lett-Brown, A. and Grant, J. A. (1979) J. lmmunol. 123, 626-632. [20] Schulman, E. S., McGettigan, M. C., Post, T. J., Vigderman, R. J. and Shapiro, S. S. (1988) J. immunol. 140, 2369- 2375. [21] O'Donnell, M. C., Ackerman, S. J., Gleich, G. J. and Thomas, L. L. (1983) J. Exp. Med. 157, 1981-1991. [22] Orchard, M. A., Kagey-Sobotka, A., Proud, D. and Lichtenstein, L. M. (1986) J. Immunol. 136, 2240-2244. [23] MacDonald, S. M., Lichtenstein, L. M., Proud, D., Plaut, M., Naclerio, R. M., MacGlashlan, D. W. and KageySobotka, A. (1987) J. lmmunol. 139, 506-512. [24] Baeza, M. L., Haak-Freridscho, M., Satnik, S. and Kaplan, A . P . J . Immunol. In p~ess. [25] Wedmore, C. V. and Williams, T. J. (1981) Nature 289, 646-650.
Incubation conditions
Net °7o histamine release
0°C 37°C
1 _+ 1.5% 14 _+ 0%
3mMCa ++,2mM Mg+ +, a 0.01 M EDTA b
16 + 3O7o 5 +_ 0.5°70
a 1.5 mM free Ca++; b 0.0001 mM free Ca +"
c u b a t e d w i t h N A P - I at 37 ° C released 14°70 o f total cell h i s t a m i n e (n = 4). T h e n e t h i s t a m i n e release o f 16°70 i n d u c e d by N A P - 1 was s i g n i f i c a n t l y lower t h a n t h e 35°7o ind u c e d b y a n t i - I g E . N A P - 1 m a y act o n a s u b p o p u l a tion of human basophils, a possibility strengthened b y t h e fact t h a t o n l y 9°7o o f h u m a n b a s o p h i l s m a d e a c h e m o t a c t i c r e s p o n s e to N A P - 1 . A g o n i s t - s p e c i f i c s u b p o p u l a t i o n s c o u l d also a c c o u n t for t h e results o f S i r a g a n i a n a n d H o o k [14], w h o s h o w e d t h a t t h e h i s t a m i n e - r e l e a s i n g effect o f 2 d i f f e r e n t secretag o g u e s a d d e d s i m u l t a n e o u s l y (e.g. a n t i - I g E + f M e t P h e - M e t o r C 5 a + f M e t - P h e - M e t ) was c o n s i d e r a b l y greater t h a n t h e effect o f either o n e alone. T h e p o s s i b i l i t y c o u l d b e tested b y flow c y t o m e t r y w i t h appropriately labeled ligands. N A P - 1 has p r e v i o u s l y b e e n c h a r a c t e r i z e d by its P M N c h e m o t a c t i c a n d a c t i v a t i n g properties. It also a t t r a c t s h u m a n b a s o p h i l s ( L e o n a r d et al., u n p u b lished), a n d , as s h o w n in this c o m m u n i c a t i o n , causes b a s o p h i l h i s t a m i n e release. I n these respects it is s i m i l a r to C5a, w h i c h also h a s c h e m o t a c t i c [9] a n d s e c r e t a g o g u e [10] a c t i v i t y for h u m a n b a s o p h i l s . A f a m i l y o f h i s t a m i n e releasing factors o r i g i n a t i n g f r o m v a r i o u s leukocytes, platelets, a n d e n d o t h e l i a l cells, as well as h u m a n n a s a l w a s h i n g s h a s r e c e n t l y b e e n d e s c r i b e d [11, 12, 18-24]. W e d m o r e a n d W i l l i a m s [25] have reviewed e v i d e n c e t h a t e x u d a t e form a t i o n requires t h e i n t e r a c t i o n o f v a s o d i l a t o r s , v a s o p e r m e a b i l i t y factors a n d c h e m o a t t r a c t a n t s . It is p o s s i b l e t h a t N A P - 1 a n d o t h e r h i s t a m i n e releasing factors act to r e c r u i t b a s o p h i l o r m a s t cell p a r t i c i p a tion in inflammation.
154
Neutrophil attractant/activation protein-1 (NAP-l) causes human basophil histamine release M a r t h a V. W h i t e 1, Teizo Y o s h i m u r a 3, William H o o k 2, M i c h a e l A. Kaliner I a n d E d w a r d J. Leonard 3 1National Institute of Allergy and Infectious Diseases, Building 10, Room 11C205, Bethesda, Maryland 20892, U.S.A.; 2National Institute of Dental Research, Building 10, Room 1A26, Bethesda, Maryland 20892, U.S.A.; 3National Cancer Institute-FCRF, Building 560, Room 12-71, Frederick, Maryland 21701, U.S.A. (Received 9 January 1989; revision received 24 April 1989; accepted 9 May 1989)
1. Summary
Basophils from five of six human donors released histamine in response to neutrophil attractant/activation protein-1 (NAP-l). Histamine release by this protein was concentration-dependent over the range of 3 x 10-7 M to 4 x 10-6 M. At 4 x 10-6 M, the mean agonist-induced release was 16_+3% (SEM) of total basophil histamine. For the same basophil preparations, release by anti-IgE was 35+6°7o. The chemotactic protein did not cause release of histamine from basophils at 0°C or in the presence of 10 mM EDTA. The time-course of histamine release was rapid; release was 43% o f maximal after 30 s and maximal after 1 min of incubation. Thus, in addition to its previously characterized neutrophil chemotactic and activating properties, this protein activates human basophils. 2. Introduction
The isolation and purification to homogeneity of
a human monocyte derived neutrophil attractant/activation protein (NAP-l) has recently been described [1-4]. Pure NAP-I can now be obtained from culture fluid of LPS-stimulated mononuclear cells in a 2-step procedure by affinity chromatography on a column of mouse monoclonal anti-NAP- 1, followed by C M - H P L C [5]. The cDNA for NAP-1 contains a coding sequence of 99 residues [6], the last 72 of which correspond to NAP- 1. Two more NAP-1 proteins, with 5 and 7 additional amino acids at the N-terminus, have been reported [5]. NAP-1 is not only a neutrophil chemoattractant, but also a secretory and metabolic stimulus, as shown by lysosomal enzyme release, superoxide and hydrogen peroxide production, and a rise in intracellular calcium ion [3, 7, 8]. Although NAP-1 has no chemotactic activity for monocytes or eosinophils, it attracts a subpopulation of human basophils (E. J. L., unpublished). This evidence for NAP-l-basophil interaction suggested that NAP-1 might induce basophil histamine release, especially in view of the fact that another protein, C5a, is both a chemoattractant [9] and a secretagogue [10] for human basophils.
Key words: Neutrophil attractant/activation protein-I; Histamine release; Basophils; Monocyte; Neutrophil; Chemotaxis
3. Materials and methods
Abbreviations." HBSS, Hanks balanced salt solution; HRA-N, neutrophil-derived histamine-releasing activity; NAP-l, neutrophil attractant activation protein; PMN, polymorphonuclear leukocyte.
3.1. NAP-1 production and isolation
Correspondence to." Martha V. White, National Institutes of Health, Building 10, Room 11C207, Bethesda, Maryland 20892, U.S.A.
NAP-1 was produced by LPS-stimulated human blood mononuclear cells and purified to homogeneity by'ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography [2]. The pure protein was stored in
0165-2478 / 89 / $ 3.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
151
35% acetonitrile at -80 °C. Prior to use, an aliquot was lyophilized and reconstituted in HBSS at a concentration of 6 x 10-6 M. Recombinant N A P - l , expressed in Escherichia coli, was supplied at a concentration of 1 m g / m l in 0.15 M NaCI by Dr. Kouji Matsushima, National Cancer Institute, Frederick, Maryland. 3.2. Preparation of human leukocytes Unfractionated leukocytes were obtained from venous blood of healthy volunteers by dextran sedimentation as previously described [11]. Blood was anticoagulated with EDTA and mixed with dextran 75 (Abbott Laboratories, North Chicago, IL). After an erythrocyte sedimentation period of 90 min, supernatant leukocytes were washed extensively in HBSS without calcium or magnesium. Just prior to histamine release experiments, cells were diluted in HBSS containing 3 m M Ca ÷ ÷ and 2 m M Mg ÷ ÷. In one experiment, basophils were separated from neutrophils by centrifugation of blood on density 1.077 Ficoll/Hypaque [12]. 3.3. Histamine release experiments To 12 x 75 polypropylene culture tubes were added 10/A aliquots of leukocyte suspension (20-40 ng total histamine) and 60/A of HBSS containing 3 mM Ca ++ (1.5 m M free Ca ++ by Ca ++ electrode measurement) and 2 m M Mg ÷ ÷ with or without secretagogue. After incubation at 37 °C for 45 min, the reaction was terminated by addition of 600 fzl HBSS without Ca ÷ ÷ or Mg ÷÷ . Cells were separated from incubation fluid by centrifugation at 4°C. Histamine content of cells and supernatants was determined by an automated fluorometric assay which is sensitive to 0.2 n g / m l [13]. Results are expressed as % histamine release corrected for spontaneous release (less than 5%) in the presence of buffer alone.
4. Results and discussion
NAP-1 caused histamine release by washed leukocyte preparations from 5 of 6 human donors tested (Fig. 1). At the highest concentration, net release was 16+_3% (SEM for 4 preparations). The 5 N A P - l responsive leukocyte preparations also released histamine when incubated with anti-IgE (0.1-1 #g/ml); net maximal release was 35_+6%. Anti-IgE induced 41% release from leukocytes of the single non-atopic donor that failed to release histamine in response to NAP-1. There was no relationship between the atopic status of the donors and the sensitivity of their basophils to N A P - l - i n d u c e d histamine release. Nor was there a correlation either by regression analysis or S p e a r m a n ' s rank correlation between the magnitudes of maximal response to anti-IgE and NAP-1. This result is comparable to that of Siraganian and H o o k [14], who studied histamine release by C5a, fMet-Phe-Met and anti-IgE. Magnitude of release was different for each donor and agonist, and magnitude of histamine release by one agonist did not correlate with that induced by any other agonist. The concentration range over which NAP-1 caused release of histamine was two orders of magnitude higher than the optimal N A P - 1 concentration (10 -8 M) required for basophil chemotaxis (Leonard et al., unpublished). If we as20 ¸
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3.4. Allergic status of basophil donors Donors were defined as allergic if they had seasonal asthma or rhinitis with positive prick skin tests to aeroallergens corresponding to their symptoms, or if they had anaphylactic reactions to foods to which they were skin test-positive.
152
1
2
3
-6 NAP-1 CONCENTRATION X 10
4-
M
Fig. 1. Histamine release in response to NAP- l by washed leukocytes from 2 atopic and 3 non-atopic donors. Cells were incubated 45 rain at 37 °C with various concentrations of N A P - 1 or buffer. Nine experiments were performed. Data points are means +_ SEM for 4 to 9 duplicate measurements at the indicated concentrations. The ordinate represents histamine release in the presence of agonist minus spontaneous release, as a percentage of total leukocyte histamine.
sume a minimum occupancy o f 5% of basophil NAP-1 receptors at an agonist concentration of 10-8 M, almost all receptors would be occupied in the 10-6 M concentration range that induced histamine release [15]. At least a 10-fold higher concentration of agonist for secretion than for chemotaxis was also required for neutrophil responses to fMetLeu-Phe [16]. The possibility that histamine release was caused by trace amounts of other cytokines in purified NAP-1 was addressed by assessing histamine release by 4 donors' basophils in response to recombinant NAP-1 (Fig. 2). All 4 donors' basophils responded to recombinant N A P - l , and the magnitude of histamine release was comparable to that induced by purified NAP-1. The data suggest that histamine release was caused by NAP-1 and not by contaminating cytokines. The time-course of NAP-l-induced basophil histamine release is illustrated in Fig. 3. Histamine release was 43% complete after 30 s and complete after 1 min of incubation. C5a and fMet-Phe-Met cause histamine release with an equally rapid time course [14]. This is in contrast to the time-course of basophil histamine release induced by anti-IgE, which is not complete until 20 min [17]. Several histamine releasing factors, including macrophage-
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Fig. 2. Histamine release in response to recombinant NAP-1 by washed leukocytes from 2 atopic and 2 non-atopic donors (3 of the donors were also used in Fig. 1). Cells were incubated 45 min at 37 °C with various concentrations of recombinant NAP-1 or buffer. Data points are means _+ SEM for 4 duplicate measurements at the indicated concentrations. The ordinate represents histamine release in the presence of agonist minus spontaneous release, as a percentage of total leukocyte histamine.
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MINUTES
Fig. 3. Time course of NAP-l-induced human basophil histamine release. Leukocytes were mixed with 4× 10-6 M NAP-1 at 37 °C. At different intervals, the reaction was stopped by the addition of ice cold 0.01 M EDTA. Tubes were centrifuged and net histamine release was determined. The data are expressed as mean _+ SEM for 4 experiments. Net maximal histamine release ranged from 14 to 26°7o.
derived histamine releasing factor, are thought to activate human basophils by interacting with IgE [18-20]. The time-course data suggest that NAP-1 does not work through this mechanism. Neutrophils spontaneously release a histamine releasing activity, termed HRA-N, which causes rapid release of basophil histamine [11, 12]. Since NAP1 is a PMN secretagogue, it was of interest to determine if NAP-l-induced histamine release was dependent upon or enhanced by the presence of PMN. Venous blood was processed as described in Section 3 to provide two leukocyte preparations, one of which was depleted of PMN. In experiments with leukocytes from 3 donors, 3 × 10 -6 M NAP-1 caused equivalent amounts of histamine release from both preparations of leukocytes (data not shown). Thus NAP-l-induced basophil histamine release is neither dependent upon nor enhanced by PMN, suggesting that NAP-1 acts directly to induce basophil activation rather than through HRA-N. Table 1 shows the temperature and divalent cation dependence of NAP-l-induced histamine release. Release was inhibited 750/0 in the presence of 0.01 M EDTA (4.5% compared to 16% net histamine release without EDTA, p < 0.025), indicating that N A P - I induced release rbquires divalent cations. No release occurred from basophils incubated with NAP-1 at 4 °C, whereas leukocytes from the same donors in153
TABLE 1
References
Temperature and divalent cation dependence of NAP-1-induced basophil histamine release.
[1] Yoshimura, T., Matsushima, K., Oppenheim, J. J. and Leonard, E. J. (1987) J. Immunol. 139, 788-793. [2] Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Appella E., Oppenheim, J. J. and Leonard, E. J. (1987) Proc. Natl. Acad. Sci. USA 84, 9233-9237. [3] Schroeder, J. M., Mrowietz, U., Morita, E. and Christophers, E. (1987) J. lmmunol. 139, 3474-3484. [4] Walz, A., Peveri, P., Aschauer, H. and Baggiolini, M. (1987) Biochem. Biophys. Res. Comm. 149, 755-761. [5] Yoshimura, T., Robinson, E. A., Appella, E., Matst/shima, K., Showalter, S. D., Skeel, A. and Leonard, E. J. (1988) Mol. lmmunol. In press. [6] Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, S. F., Leonard, E. J. and Oppenheim, J. J. (1988) J. Exp. Med. 167, 1883-1893. [7] Peveri, P., Walz, A., Dewald, B. and Baggiolini, M. (1988) J. Exp. Med. 167, 1547-1559. [8] Thelen, M., Peveri, P., Karnen, P., von Tscharner, V., Waltz, A. and Baggiolini, M. (1988) FASEB J. 2, 2702-2706. [9] Lett-Brown, M., Boetcher, D. A. and Leonard, E. J. (1976) J. lmmunol. 117, 246-252. [10] Hook, W., Siraganian, R. P. and Wahl, S. (1975) J. immunol. 114, 1185-1190. [11] White, M. V., Kaplan, A. P., Haak-Frendscho, M. and Kaliner, M. (1988) J. lmmunol. 141, 3575-3583. [12] White, M. V. and Kaliner, M. A. (1987) J. Immunol. 139, 1624-1630. [13] Siraganian, R. P. (1974) Anal. Biochem. 57, 383-394. [14] Siraganian, R. P. and Hook, W. A. (1977) J. lmmunol. 119, 2078-2083. [15] Waud, D. R. (1968) Pharmacol. Rev. 20, 49-88. [16] Snyderman, R. (1984) Federation Proc. 43, 2743-2748. [17] Gillespie, E. and Lichtenstein, L. M. (1972) J. Clin. Invest. 51, 2941-2947. [18] Liu, M. C., Proud, D., Lichtenstein, L. M., MacGlashlan Jr., D. W., Schleimer, R. P., Adkinson Jr., N. F., Kagey-Sobotka, A., Schulman, E. S. and Plaut, M. (1986) J. lmmunol. 136, 2588-2595. [19] Theuson, D. O., Speck, L. S., Lett-Brown, A. and Grant, J. A. (1979) J. lmmunol. 123, 626-632. [20] Schulman, E. S., McGettigan, M. C., Post, T. J., Vigderman, R. J. and Shapiro, S. S. (1988) J. immunol. 140, 2369- 2375. [21] O'Donnell, M. C., Ackerman, S. J., Gleich, G. J. and Thomas, L. L. (1983) J. Exp. Med. 157, 1981-1991. [22] Orchard, M. A., Kagey-Sobotka, A., Proud, D. and Lichtenstein, L. M. (1986) J. Immunol. 136, 2240-2244. [23] MacDonald, S. M., Lichtenstein, L. M., Proud, D., Plaut, M., Naclerio, R. M., MacGlashlan, D. W. and KageySobotka, A. (1987) J. lmmunol. 139, 506-512. [24] Baeza, M. L., Haak-Freridscho, M., Satnik, S. and Kaplan, A . P . J . Immunol. In p~ess. [25] Wedmore, C. V. and Williams, T. J. (1981) Nature 289, 646-650.
Incubation conditions
Net °7o histamine release
0°C 37°C
1 _+ 1.5% 14 _+ 0%
3mMCa ++,2mM Mg+ +, a 0.01 M EDTA b
16 + 3O7o 5 +_ 0.5°70
a 1.5 mM free Ca++; b 0.0001 mM free Ca +"
c u b a t e d w i t h N A P - I at 37 ° C released 14°70 o f total cell h i s t a m i n e (n = 4). T h e n e t h i s t a m i n e release o f 16°70 i n d u c e d by N A P - 1 was s i g n i f i c a n t l y lower t h a n t h e 35°7o ind u c e d b y a n t i - I g E . N A P - 1 m a y act o n a s u b p o p u l a tion of human basophils, a possibility strengthened b y t h e fact t h a t o n l y 9°7o o f h u m a n b a s o p h i l s m a d e a c h e m o t a c t i c r e s p o n s e to N A P - 1 . A g o n i s t - s p e c i f i c s u b p o p u l a t i o n s c o u l d also a c c o u n t for t h e results o f S i r a g a n i a n a n d H o o k [14], w h o s h o w e d t h a t t h e h i s t a m i n e - r e l e a s i n g effect o f 2 d i f f e r e n t secretag o g u e s a d d e d s i m u l t a n e o u s l y (e.g. a n t i - I g E + f M e t P h e - M e t o r C 5 a + f M e t - P h e - M e t ) was c o n s i d e r a b l y greater t h a n t h e effect o f either o n e alone. T h e p o s s i b i l i t y c o u l d b e tested b y flow c y t o m e t r y w i t h appropriately labeled ligands. N A P - 1 has p r e v i o u s l y b e e n c h a r a c t e r i z e d by its P M N c h e m o t a c t i c a n d a c t i v a t i n g properties. It also a t t r a c t s h u m a n b a s o p h i l s ( L e o n a r d et al., u n p u b lished), a n d , as s h o w n in this c o m m u n i c a t i o n , causes b a s o p h i l h i s t a m i n e release. I n these respects it is s i m i l a r to C5a, w h i c h also h a s c h e m o t a c t i c [9] a n d s e c r e t a g o g u e [10] a c t i v i t y for h u m a n b a s o p h i l s . A f a m i l y o f h i s t a m i n e releasing factors o r i g i n a t i n g f r o m v a r i o u s leukocytes, platelets, a n d e n d o t h e l i a l cells, as well as h u m a n n a s a l w a s h i n g s h a s r e c e n t l y b e e n d e s c r i b e d [11, 12, 18-24]. W e d m o r e a n d W i l l i a m s [25] have reviewed e v i d e n c e t h a t e x u d a t e form a t i o n requires t h e i n t e r a c t i o n o f v a s o d i l a t o r s , v a s o p e r m e a b i l i t y factors a n d c h e m o a t t r a c t a n t s . It is p o s s i b l e t h a t N A P - 1 a n d o t h e r h i s t a m i n e releasing factors act to r e c r u i t b a s o p h i l o r m a s t cell p a r t i c i p a tion in inflammation.
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