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367 The cell surface antigens CD44 and CD69 are activation markers for human eosinophils (EOS)
274
Abstracts
J ALLERGY CLIN IMMUNOL
JANUARY 1996 365
HEPARIN INHIBITS RA NTES- INDUCED CHEMOTAXIS OF EOSINOPHILS. P Kuna MD. PhD. M Antczak MD. G Cieslewic;~ MD. PhD. J Rozniecki MD. Lodz, Poland Heparin has been reported to diminish the size of allergen skin tests and decrease the response to allergen bronchoprovocation in allergic asthma. We have found that heparin diminished the late phase of the allergic reaction and inhibits the allergen induced increase in specific and nonspecific bronchial hyperresponsiveness. In order to explore the mechanism involved in these protective effects we studied the influence of heparin on eosinophil chemotaxis induced by RANTES and FMLP. Eosinophils were isolated from the peripheral blood of 12 asthmatic donors. Chemotaxis was evaluated using polycarbonate filters in 48 well chemotactic chambers. The table shows the heparin dose in units/ml and chemotactic index for inhibition of RANTES and FMLP (*)p<0.05.
367
RP Schleimer PhD. BS B~)chner ~,~D, Baltimore, MD Activated EOS are important effector cells in allergic diseases. Information regarding their function may be obtained by determining the pattern o f expression of cell surface antigens. We therefore analyzed purified peripheral blood or late phase bronchoalveolar lavage (BAL) EOS, using 473 monoclonai antibodies ( m A b s ) from the 5th International Workshop on H u m a n Leukocyte Antigens, for surface markers absent on fresh EOS but present after in vivo activation or culture for up to 72 hr with cytokines (< 10 ng/ml of IL-3, IL-5, or G M - C S F ) , or markers normally expressed on fresh eosinophils which are altered after cytokine culture. Using indirect immunofluorescence and flow cytometry, only m A b s against CD69 bound to late phase B A L or cytokine-cuhured EOS but not fresh EOS. Significant concentration- and time-dependent increases in CD44 expression were observed in EOS cultured with cytokines (~ 2-3 fold increase in fluorescence intensity, p<0.05), while expression o f other constitutively expressed markers (e.g., CD9, C D I la) was unchanged. However, neither fresh nor cultured EOS adhered to immobilized hyaluronic acid, a ligand for CD44, even though the basophilic leukemia cell line KU812 displayed C D 4 4 - d e p e n d e n t a d h e s i o n to h y a l u r o n i c acid. We conclude that activation of EOS in vitro or in vivo leads to induction or increased expression o f CD44 and CD69. The functional significance of these changes remains unknown.
0.0 IU 10 IU 25IU 50IU 100IU RANTES 3.5:L'0.6 2.6:L,0.7" 1.2_.+0.35" 0.74-0.4* 2.7+0.9 FMLP 6.2+2.1 2.05:0.7* 1.64-0.35" 2.54-0.8* 3.0:L-0.3" Ten minutes of exposure of eosinophils to heparin was sufficient for inhibition. Removal of heparin by washing the cells ablated the observed effects. Preincubation of RANTES or FMLP with heparin did not induce any significant changes in eosinophils chemotaxis. Heparin alone did not possess any chemotactic activity for eosinophils and did not induce intracellular calcium flux. Our results suggest that heparin ameliorates allergic inflammation by inhibition of eosinophil flux and this inhibition is a surface phenomenon.
366
Injection
of Histamine with Mast Cell Tryptase Selectively Stimulates Eosinophil Accumulation In Vivo.
S He MD. O Pena BM. AF Walls PhD. Southampton, UK Tryptase, the most abundant protein constituent of human mast cells, is secreted from these cells together with histamine, heparin and other preformed mediators in allergic disease. In order to investigate the actions of this protease in rive, we have injected purified preparations of human tryptase (5 ng to 50 lag)in the presence or absence of other mast cell products into the skin of guinea pigs and the peritoneum of mice. After 3h, 6 h or 16 h animals were killed and ceils enumerated in tissue at the injection site in skin or.in peritoneal lavage fluid. In both models, injection of tryptase alone resulted in significant increases in numbers of neutrophils at all time points and in eosinophils at 6 h and 16 h. At the later time points there were small but significant elevations in macrophage and lymphocyte numbers in peritoneal lavage fluid. Coinjection of protease inhibitors with tryptase reduced cell numbers indicating dependence on the catalytic site. Injection ofheparin alone over a similar range of concentrations did not stimulate cell recruitment and co-injection with tryptase did not significantly alter the response to tryptase. On the other hand, though histamine by itself did not induce cell accumulation, when added to tryptase over several concentrations, there were selective increases in eosinophil numbers of up to 10 fold, as well as small but significant increases in numbers of lymphocytes. The release of tryptase with histamine from activated mast cells may be important in stimulating the accumulation of eosinophils and other inflammatory cells in allergic disease.
The cell surface antigens CD44 and CD69 are a c t i v a t i o n markers for human eosinophils (EOS). K Matsumoto MD. J ADoiah-PiDnim MD. C Bickel MS.
368
The differential expression of adhesion molecules on airway and circulating eosinophils DA WongMD and PM O'Bvme M D Asthma Research Group, McMaster University, Hamilton, ON, C A N A D A Adhesion molecules play a role in the selectivemigration ofleukocytes and theirregulation.To study which adhesion molecules are involved in
the eosinophil we examined the changes in adhesion molecule expression as eosinophils moved from the circulation into the airway. Depolarized light was used to identify eosinophils by flow cytometry allowing the quantitative study of cell surface proteins on ceils in blood and the airway of humans and dogs. Blood and induced sputum from subjects with mild asthma, not on regular treatment; or blood and bronchoalveolar lavage from dogs were collected, and leukocytes were then stained with a panel of adhesion antibodies. Circulating eosinophils were found to express ¢x4~,(CD49d/CD29),~413~(CD49d/[37),~L[32(CD11a/CD18),L-selectin (CD62L), and ICAM-3(CD50). Sputum eosinophils showed a decreased level of expression of L-selectin and ICAM-3 while there was increased expression 132integrins, [CtL,aM(CD11b),ctX(CD11c)] and ICAM-1(CD54). In particular, in humans there is little to no expression of ~x in blood while it is expressed on sputum eosinophils. In dog, cxx is expressed on circulating eosinophils but this increases after migration into the airway. The binding of an integrin to its ligand transduces an activating signal to the cell; thus suggesting a role ~32integrins and ICAM-1 in cell movement and regulation after the eosinophil after leaves the blood stream. These results demonstrate that the expression of adhesion proteins on the surface of dog and human eosinophils changes as a cell moves into the airway. Improved understanding of how adhesion molecules are modified during migration and activation ofeosinophils may allow us to targeting specific inhibitors (for example to c~x) in the future.
Abstracts
J ALLERGY CLIN IMMUNOL
JANUARY 1996 365
HEPARIN INHIBITS RA NTES- INDUCED CHEMOTAXIS OF EOSINOPHILS. P Kuna MD. PhD. M Antczak MD. G Cieslewic;~ MD. PhD. J Rozniecki MD. Lodz, Poland Heparin has been reported to diminish the size of allergen skin tests and decrease the response to allergen bronchoprovocation in allergic asthma. We have found that heparin diminished the late phase of the allergic reaction and inhibits the allergen induced increase in specific and nonspecific bronchial hyperresponsiveness. In order to explore the mechanism involved in these protective effects we studied the influence of heparin on eosinophil chemotaxis induced by RANTES and FMLP. Eosinophils were isolated from the peripheral blood of 12 asthmatic donors. Chemotaxis was evaluated using polycarbonate filters in 48 well chemotactic chambers. The table shows the heparin dose in units/ml and chemotactic index for inhibition of RANTES and FMLP (*)p<0.05.
367
RP Schleimer PhD. BS B~)chner ~,~D, Baltimore, MD Activated EOS are important effector cells in allergic diseases. Information regarding their function may be obtained by determining the pattern o f expression of cell surface antigens. We therefore analyzed purified peripheral blood or late phase bronchoalveolar lavage (BAL) EOS, using 473 monoclonai antibodies ( m A b s ) from the 5th International Workshop on H u m a n Leukocyte Antigens, for surface markers absent on fresh EOS but present after in vivo activation or culture for up to 72 hr with cytokines (< 10 ng/ml of IL-3, IL-5, or G M - C S F ) , or markers normally expressed on fresh eosinophils which are altered after cytokine culture. Using indirect immunofluorescence and flow cytometry, only m A b s against CD69 bound to late phase B A L or cytokine-cuhured EOS but not fresh EOS. Significant concentration- and time-dependent increases in CD44 expression were observed in EOS cultured with cytokines (~ 2-3 fold increase in fluorescence intensity, p<0.05), while expression o f other constitutively expressed markers (e.g., CD9, C D I la) was unchanged. However, neither fresh nor cultured EOS adhered to immobilized hyaluronic acid, a ligand for CD44, even though the basophilic leukemia cell line KU812 displayed C D 4 4 - d e p e n d e n t a d h e s i o n to h y a l u r o n i c acid. We conclude that activation of EOS in vitro or in vivo leads to induction or increased expression o f CD44 and CD69. The functional significance of these changes remains unknown.
0.0 IU 10 IU 25IU 50IU 100IU RANTES 3.5:L'0.6 2.6:L,0.7" 1.2_.+0.35" 0.74-0.4* 2.7+0.9 FMLP 6.2+2.1 2.05:0.7* 1.64-0.35" 2.54-0.8* 3.0:L-0.3" Ten minutes of exposure of eosinophils to heparin was sufficient for inhibition. Removal of heparin by washing the cells ablated the observed effects. Preincubation of RANTES or FMLP with heparin did not induce any significant changes in eosinophils chemotaxis. Heparin alone did not possess any chemotactic activity for eosinophils and did not induce intracellular calcium flux. Our results suggest that heparin ameliorates allergic inflammation by inhibition of eosinophil flux and this inhibition is a surface phenomenon.
366
Injection
of Histamine with Mast Cell Tryptase Selectively Stimulates Eosinophil Accumulation In Vivo.
S He MD. O Pena BM. AF Walls PhD. Southampton, UK Tryptase, the most abundant protein constituent of human mast cells, is secreted from these cells together with histamine, heparin and other preformed mediators in allergic disease. In order to investigate the actions of this protease in rive, we have injected purified preparations of human tryptase (5 ng to 50 lag)in the presence or absence of other mast cell products into the skin of guinea pigs and the peritoneum of mice. After 3h, 6 h or 16 h animals were killed and ceils enumerated in tissue at the injection site in skin or.in peritoneal lavage fluid. In both models, injection of tryptase alone resulted in significant increases in numbers of neutrophils at all time points and in eosinophils at 6 h and 16 h. At the later time points there were small but significant elevations in macrophage and lymphocyte numbers in peritoneal lavage fluid. Coinjection of protease inhibitors with tryptase reduced cell numbers indicating dependence on the catalytic site. Injection ofheparin alone over a similar range of concentrations did not stimulate cell recruitment and co-injection with tryptase did not significantly alter the response to tryptase. On the other hand, though histamine by itself did not induce cell accumulation, when added to tryptase over several concentrations, there were selective increases in eosinophil numbers of up to 10 fold, as well as small but significant increases in numbers of lymphocytes. The release of tryptase with histamine from activated mast cells may be important in stimulating the accumulation of eosinophils and other inflammatory cells in allergic disease.
The cell surface antigens CD44 and CD69 are a c t i v a t i o n markers for human eosinophils (EOS). K Matsumoto MD. J ADoiah-PiDnim MD. C Bickel MS.
368
The differential expression of adhesion molecules on airway and circulating eosinophils DA WongMD and PM O'Bvme M D Asthma Research Group, McMaster University, Hamilton, ON, C A N A D A Adhesion molecules play a role in the selectivemigration ofleukocytes and theirregulation.To study which adhesion molecules are involved in
the eosinophil we examined the changes in adhesion molecule expression as eosinophils moved from the circulation into the airway. Depolarized light was used to identify eosinophils by flow cytometry allowing the quantitative study of cell surface proteins on ceils in blood and the airway of humans and dogs. Blood and induced sputum from subjects with mild asthma, not on regular treatment; or blood and bronchoalveolar lavage from dogs were collected, and leukocytes were then stained with a panel of adhesion antibodies. Circulating eosinophils were found to express ¢x4~,(CD49d/CD29),~413~(CD49d/[37),~L[32(CD11a/CD18),L-selectin (CD62L), and ICAM-3(CD50). Sputum eosinophils showed a decreased level of expression of L-selectin and ICAM-3 while there was increased expression 132integrins, [CtL,aM(CD11b),ctX(CD11c)] and ICAM-1(CD54). In particular, in humans there is little to no expression of ~x in blood while it is expressed on sputum eosinophils. In dog, cxx is expressed on circulating eosinophils but this increases after migration into the airway. The binding of an integrin to its ligand transduces an activating signal to the cell; thus suggesting a role ~32integrins and ICAM-1 in cell movement and regulation after the eosinophil after leaves the blood stream. These results demonstrate that the expression of adhesion proteins on the surface of dog and human eosinophils changes as a cell moves into the airway. Improved understanding of how adhesion molecules are modified during migration and activation ofeosinophils may allow us to targeting specific inhibitors (for example to c~x) in the future.