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A chemotaxis assay using human polymorphonuclear leukocytes stimulated by IL-1
Journal of Immunological Methods, 112 (1988) 145-146
145
Elsevier JIM 04908
Short communication
A chemotaxis assay using human polymorphonuclear leukocytes stimulated by IL-1 Bruce L. Maloff, Joan E. Shaw and Diane Fox E.I. Du Pont de Nemours & Co., Inc., Experimental Station, Medical Products Department, Immunopharmacology Section, Wilmington, DE 19898, U.S.A.
(Received 22 March 1988, accepted 11 May 1988)
Key words: Interleukin-1, Chemotaxis; Neutrophil
Interleukin-1 (IL-1) is a polypeptide hormone, p r o d u c e d b y m o n o n u c l e a r phagocytes, which m a y play a role in the pathogenesis of several imm u n o i n f l a m m a t o r y diseases. IL-1 has been reported to stimulate a n u m b e r of bioresponses associated with inflammation in h u m a n polymorphonuclear leukocytes, including release of granule-associated enzymes and production of LTB 4 (Smith et al., 1987). The ability of neutrophils to c h e m o t a x to IL-1 is currently unresolved; Westcott et al. (1987) have reported chemotactic activity of IL-1, whereas Georgilis et al. (1987) found that IL-1 was not chemotactic. This report describes the dependence on the concentration of bovine serum albumin in the m e d i u m for chemotaxis of h u m a n neutrophils to IL-1. H u m a n p o l y m o r p h o n u c l e a r leukocytes were prepared b y standard techniques as have been previously described (Maloff et al., 1987). 2 × 10 s cells were aliquoted into the upper well of a B o y d e n c h a m b e r and separated by filter paper f r o m the chemoattractant placed in the lower well
Correspondence to: B.L. Maloff, E.I. Du Pont de Nemours&Co., Inc., Experimental Station, Medical Products Department, Immunopharmacology Section, Wilmington, DE 19898, U.S.A.
of the c h a m b e r (Boyden, 1962). Cells and c h e m o attractant were maintained in H a n k s ' balanced salt solution with the concentrations indicated of BSA. Following 90 min of incubation at 37 o C,
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Fig. 1. Effect of LTB4, FMLP, and IL-1 on chemotaxis of human neutrophils. Cells were incubated for 90 min in the presence of chemotactic stimuli, filter paper stained with hematoxylin, and movement quantitated by measuring distance to the leading front. Data are expressed as mean + SEM
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
( n = 5).
146 8O
6O
! 4o
0.1
0.2 1 2 */.BSA in medium Fig. 2. Effect of BSA on chemotaxis. Assay conditions were the same as those in Fig. 1 with the BSA concentration altered as indicated for the various stimuli; IL-I(100 U / m l ) (open bars), LTB4(1 x 10 8 M) (hatched bars), or FMLP(1 X 10 -8 M) (solid bars). Data are expressed as m e a n + SEM (n = 5).
filters were removed, stained with hematoxylin, and mounted on slides. Cell migration was quantitated by measuring the distance from the primary plane of cells to the leading front. Chemotaxis was evaluated in response to maximally effective concentrations of two well-established chemoattractants, leukotriene B4 and the synthetic oligopeptide, formyl-methionyl-leucylphenylalanine, and to varying concentrations of IL-1 (Fig. 1). The IL-1 used was rlL-lfl prepared by Dr. James H u a n g of the Du Pont Company. IL-1 produced a dose-dependent chemotactic response at 1% BSA, which reaches a maximal effect at 100 U / m l IL-1. Although the efficacy of IL-1 to elicit a chemotaxis of h u m a n neutrophils is about one-half that of the standard chemoattractants, it is an order of magnitude more potent. In the study of Georgilis et al. in which IL-1 was not chemotactic, a 2% concentration of BSA was used; in the present study, a final concentration of 1% BSA was employed in the standard
assays. In this regard, a dose-response to BSA was investigated to establish the potential role of this protein buffer on biologic response to IL-1 (Fig. 2). A minimal level of BSA, between 0.2 and 1.0%, was required to elicit a chemotactic response for any of the stimuli. At 2% BSA, however, a marked diminution of the LTB 4 response and complete elimination of the IL-1 response was observed. This is likely due to the ability of BSA to act as a molecular scavenger. At the concentration of BSA used in the Georgilis study, IL-1 was effectively reduced to a non-stimulatory concentration. It is interesting that the oligopeptide F M L P , which is less likely to be bound by BSA than LTB 4 or IL-1, maintained efficacy at the higher BSA concentration. This study illustrates the conditions under which IL-1 can be used as a chemoattractant for h u m a n neutrophils. Inhibition of the signalling of IL-1 may provide a novel, disease-modifying pharmacotherapy for a number of inflammatory conditions. Measurement of IL-1 chemotaxis in these clinically relevant cells may be a useful tool for identifying potential IL-1 antagonists.
References Boyden, S. (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453. Georgilis, K., Schaefer, C., Dinarello, C.A. and Klempner, M.S. (1987) H u m a n recombinant interleukin 1 beta has no effect on intraceUular calcium or on functional responses of h u m a n neutrophils. J. Immunol. 138, 3403. Maloff, B., Fefer, D., Cooke, G.M. and Ackerman, N.R. (1987) Inhibition of LTB 4 binding to h u m a n neutrophils by nordihydroguaiaretic acid. Agents Actions 21, 358. Smith, R.J., Bowman, B.J. and Speziale, S.C. (1985) Interleukin-1 stimulates granule exocytosis from h u m a n neutrophils. Int. J. Immunopharmacol. 8, 33. Westmacott, D., Wadsworth, J. and Bloxham, D.P. (1987) Chemotactic activity of recombinant interleukin-1. Agents Actions 21,323.
145
Elsevier JIM 04908
Short communication
A chemotaxis assay using human polymorphonuclear leukocytes stimulated by IL-1 Bruce L. Maloff, Joan E. Shaw and Diane Fox E.I. Du Pont de Nemours & Co., Inc., Experimental Station, Medical Products Department, Immunopharmacology Section, Wilmington, DE 19898, U.S.A.
(Received 22 March 1988, accepted 11 May 1988)
Key words: Interleukin-1, Chemotaxis; Neutrophil
Interleukin-1 (IL-1) is a polypeptide hormone, p r o d u c e d b y m o n o n u c l e a r phagocytes, which m a y play a role in the pathogenesis of several imm u n o i n f l a m m a t o r y diseases. IL-1 has been reported to stimulate a n u m b e r of bioresponses associated with inflammation in h u m a n polymorphonuclear leukocytes, including release of granule-associated enzymes and production of LTB 4 (Smith et al., 1987). The ability of neutrophils to c h e m o t a x to IL-1 is currently unresolved; Westcott et al. (1987) have reported chemotactic activity of IL-1, whereas Georgilis et al. (1987) found that IL-1 was not chemotactic. This report describes the dependence on the concentration of bovine serum albumin in the m e d i u m for chemotaxis of h u m a n neutrophils to IL-1. H u m a n p o l y m o r p h o n u c l e a r leukocytes were prepared b y standard techniques as have been previously described (Maloff et al., 1987). 2 × 10 s cells were aliquoted into the upper well of a B o y d e n c h a m b e r and separated by filter paper f r o m the chemoattractant placed in the lower well
Correspondence to: B.L. Maloff, E.I. Du Pont de Nemours&Co., Inc., Experimental Station, Medical Products Department, Immunopharmacology Section, Wilmington, DE 19898, U.S.A.
of the c h a m b e r (Boyden, 1962). Cells and c h e m o attractant were maintained in H a n k s ' balanced salt solution with the concentrations indicated of BSA. Following 90 min of incubation at 37 o C,
80
Y/////
60
E o
4C
"////,ff ~////.6, "/'////G "/'////~
//'///.4, V///..~
v//////
o y
2C
U/////, ~///111 ~'////% "/////% ///I/I/ I////// I
I
d
"
I
I
"7
"7
I
Fig. 1. Effect of LTB4, FMLP, and IL-1 on chemotaxis of human neutrophils. Cells were incubated for 90 min in the presence of chemotactic stimuli, filter paper stained with hematoxylin, and movement quantitated by measuring distance to the leading front. Data are expressed as mean + SEM
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
( n = 5).
146 8O
6O
! 4o
0.1
0.2 1 2 */.BSA in medium Fig. 2. Effect of BSA on chemotaxis. Assay conditions were the same as those in Fig. 1 with the BSA concentration altered as indicated for the various stimuli; IL-I(100 U / m l ) (open bars), LTB4(1 x 10 8 M) (hatched bars), or FMLP(1 X 10 -8 M) (solid bars). Data are expressed as m e a n + SEM (n = 5).
filters were removed, stained with hematoxylin, and mounted on slides. Cell migration was quantitated by measuring the distance from the primary plane of cells to the leading front. Chemotaxis was evaluated in response to maximally effective concentrations of two well-established chemoattractants, leukotriene B4 and the synthetic oligopeptide, formyl-methionyl-leucylphenylalanine, and to varying concentrations of IL-1 (Fig. 1). The IL-1 used was rlL-lfl prepared by Dr. James H u a n g of the Du Pont Company. IL-1 produced a dose-dependent chemotactic response at 1% BSA, which reaches a maximal effect at 100 U / m l IL-1. Although the efficacy of IL-1 to elicit a chemotaxis of h u m a n neutrophils is about one-half that of the standard chemoattractants, it is an order of magnitude more potent. In the study of Georgilis et al. in which IL-1 was not chemotactic, a 2% concentration of BSA was used; in the present study, a final concentration of 1% BSA was employed in the standard
assays. In this regard, a dose-response to BSA was investigated to establish the potential role of this protein buffer on biologic response to IL-1 (Fig. 2). A minimal level of BSA, between 0.2 and 1.0%, was required to elicit a chemotactic response for any of the stimuli. At 2% BSA, however, a marked diminution of the LTB 4 response and complete elimination of the IL-1 response was observed. This is likely due to the ability of BSA to act as a molecular scavenger. At the concentration of BSA used in the Georgilis study, IL-1 was effectively reduced to a non-stimulatory concentration. It is interesting that the oligopeptide F M L P , which is less likely to be bound by BSA than LTB 4 or IL-1, maintained efficacy at the higher BSA concentration. This study illustrates the conditions under which IL-1 can be used as a chemoattractant for h u m a n neutrophils. Inhibition of the signalling of IL-1 may provide a novel, disease-modifying pharmacotherapy for a number of inflammatory conditions. Measurement of IL-1 chemotaxis in these clinically relevant cells may be a useful tool for identifying potential IL-1 antagonists.
References Boyden, S. (1962) The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leukocytes. J. Exp. Med. 115, 453. Georgilis, K., Schaefer, C., Dinarello, C.A. and Klempner, M.S. (1987) H u m a n recombinant interleukin 1 beta has no effect on intraceUular calcium or on functional responses of h u m a n neutrophils. J. Immunol. 138, 3403. Maloff, B., Fefer, D., Cooke, G.M. and Ackerman, N.R. (1987) Inhibition of LTB 4 binding to h u m a n neutrophils by nordihydroguaiaretic acid. Agents Actions 21, 358. Smith, R.J., Bowman, B.J. and Speziale, S.C. (1985) Interleukin-1 stimulates granule exocytosis from h u m a n neutrophils. Int. J. Immunopharmacol. 8, 33. Westmacott, D., Wadsworth, J. and Bloxham, D.P. (1987) Chemotactic activity of recombinant interleukin-1. Agents Actions 21,323.