Copyright 0 1979by Academic Press, hc. All rights cf reproduction in any form reserved 0014-4827/79/090169-0~02.00/0
Experimental
THE EFFECTS ADHESIVENESS RONALD
Cell Research 122 (1979) 169-177
OF CHEMOTACTIC OF RABBIT
P. C. SMITH,’
FACTORS
NEUTROPHIL
JOHN M. LACKIE’
ON THE
GRANULOCUT
and PETER C. WILKINSON”
‘Department of Cell Biology, University of Glasgow, Glasgow G12 SQQ and ‘Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow Gli 6NT, UK
SUMMARY A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, cYs-casein,p-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of Lu,-caseinand formyl-methionylleucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10-8-10-7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.
During an acute inflammatory response, neutrophil granulocytes adhere to the endothelial cells which line the post-capillary venules. Since neutrophils do not normally adhere to blood vessel walls in large numbers, their localized adhesion in areas of inflammation suggests that a change in the strength of the neutrophil-endothehal interaction may occur. The cellular location of this change is not clear but evidence supports the involvement of both the endothelium and the neutrophils [ 11. Following adhesion to the endothelium, neutrophils migrate between the endothelial cells and towards the site of damage or infection. The direction of migration is probably influenced by diffusible chemotactic factors, although the evidence for this in
vivo, is largely circumstantial [2]. Adhesion is an essential prerequisite for cell movement, and the adhesiveness of the substratum may affect both the rate and direction of locomotion; this has been shown for fibroblasts [3, 4, 51 and is probably also t for neutrophils [6]. Thus, adhesion changes might be important during inflammation not only in the primary neutrophil-endothelial interaction, but also in the chemotacticahy guided migration of neutrophils. If chemotactic factors affect the adhesive properties of neutrophils then the two phases of a hesion and migration could be mediated by a common factor. Quantitative data on adhesion are very difficult and laborious to obtain in vivo. Accordingly, we have studied the effect on neutrophil adhesion of Exp Cell Rer 122 (197Yj
170
Smith, Lackie and Wilkinson
Table 1. Effect of chemotactic factors on the adhesion after 30 min at 37°C of neutrophil granulocytes to confluent endothelium and serum-coated glass % adhesion (compared to controls= 100%) k S.E.M. (n) Chemotactic factor a,-Casein p-Casein Alkali-denatured HSA Native HSA” f-tri-tyr f-met-leu-phe f-met-phe met-pheb
Cone Of)
Endothelium
Serum-coated glass
43i5 (6) 64+4 (i3j 57k8 (12)
625 3 (3)* 49+ 6 (6) 25f 6 (3)*
136i7 (14) 6327 (11)
143&10 (8)
4.2x 1O-5 4.2x 1O-5
1.4x 10-5 1.4x10-5 10-g 10-s 10-S 10-S
46f4 (22) 43+3 (30) 84+6(13)**
6Ok 592 55+ 83i
5 (16) 5 (23) 4(33) 5 (13)*
n, no. of replicate coverslips. Using Student’s r-test, p (difference from controls=O)
Experimental
THE EFFECTS ADHESIVENESS RONALD
Cell Research 122 (1979) 169-177
OF CHEMOTACTIC OF RABBIT
P. C. SMITH,’
FACTORS
NEUTROPHIL
JOHN M. LACKIE’
ON THE
GRANULOCUT
and PETER C. WILKINSON”
‘Department of Cell Biology, University of Glasgow, Glasgow G12 SQQ and ‘Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow Gli 6NT, UK
SUMMARY A range of chemotactic factors has been shown to affect the adhesion of rabbit peritoneal neutrophil granulocytes to cultured endothelial cells and to serum-coated glass. At chemotactically optimal concentrations, cYs-casein,p-casein, alkali denatured human serum albumin (HSA) and several synthetic formyl-peptides reduced the number of adherent neutrophils after 30 min to around 50% of control values. These effects were still observed after neutrophils, but not endothelium or serum-coated glass had been exposed to chemotactic factors and washed before use in assays. Two non-chemotactic analogues, native HSA and a non-formyl-peptide were ineffective. The dose responses for adhesion after 30 min in the presence of Lu,-caseinand formyl-methionylleucyl-phenylalanine (FMLP) were found to be inversely related to those for migration towards these substances. After incubation for 60 min in high (10-8-10-7 M) concentrations of FMLP, neutrophil adhesion was found to be enhanced. Neutrophil aggregation was also affected by the presence of chemotactic factors in a similar time- and dose-dependent manner to the adhesion to substratum assays. Using FMLP, it was also shown that the timing of the adhesive changes depended on the concentration of chemotactic factor present.
During an acute inflammatory response, neutrophil granulocytes adhere to the endothelial cells which line the post-capillary venules. Since neutrophils do not normally adhere to blood vessel walls in large numbers, their localized adhesion in areas of inflammation suggests that a change in the strength of the neutrophil-endothehal interaction may occur. The cellular location of this change is not clear but evidence supports the involvement of both the endothelium and the neutrophils [ 11. Following adhesion to the endothelium, neutrophils migrate between the endothelial cells and towards the site of damage or infection. The direction of migration is probably influenced by diffusible chemotactic factors, although the evidence for this in
vivo, is largely circumstantial [2]. Adhesion is an essential prerequisite for cell movement, and the adhesiveness of the substratum may affect both the rate and direction of locomotion; this has been shown for fibroblasts [3, 4, 51 and is probably also t for neutrophils [6]. Thus, adhesion changes might be important during inflammation not only in the primary neutrophil-endothelial interaction, but also in the chemotacticahy guided migration of neutrophils. If chemotactic factors affect the adhesive properties of neutrophils then the two phases of a hesion and migration could be mediated by a common factor. Quantitative data on adhesion are very difficult and laborious to obtain in vivo. Accordingly, we have studied the effect on neutrophil adhesion of Exp Cell Rer 122 (197Yj
170
Smith, Lackie and Wilkinson
Table 1. Effect of chemotactic factors on the adhesion after 30 min at 37°C of neutrophil granulocytes to confluent endothelium and serum-coated glass % adhesion (compared to controls= 100%) k S.E.M. (n) Chemotactic factor a,-Casein p-Casein Alkali-denatured HSA Native HSA” f-tri-tyr f-met-leu-phe f-met-phe met-pheb
Cone Of)
Endothelium
Serum-coated glass
43i5 (6) 64+4 (i3j 57k8 (12)
625 3 (3)* 49+ 6 (6) 25f 6 (3)*
136i7 (14) 6327 (11)
143&10 (8)
4.2x 1O-5 4.2x 1O-5
1.4x 10-5 1.4x10-5 10-g 10-s 10-S 10-S
46f4 (22) 43+3 (30) 84+6(13)**
6Ok 592 55+ 83i
5 (16) 5 (23) 4(33) 5 (13)*
n, no. of replicate coverslips. Using Student’s r-test, p (difference from controls=O)
