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THE CHEMILUMINESCENCE OF OVINE NEUTROPHILS
A the s i s presented in partial fulfilment o f the requirement for the degree of
Master of Veterinary Sc ience at Massey University
Jane Eagleson November 1 984
ABSTRACT
The development, structure and function of polymorphonuclear leucocytes (PMN) is reviewed and methods for determining neutrophil competence are discussed.
A technique, based on differential centrifugation and red blood cell lysis, is described for isolating neutrophils of 80 to 90%
purity from ovine blood. A standardised, luminol-enhanced chemiluminescence (CL) assay was developed for ovine neutrophils using latex beads as the phagocytic stimulus and some conditions influencing the level of CL generated are described.
Normal sheep of similar age, housed under identical conditions and bled at approximately the same time on different days produced CL responses ranging from 386 to 3084 millivolts (mV). Animals sampled once daily over 5 days showed large fluctuations in CL values both between and within individuals. Furthermore, sheep bled at 4 hourly and 6 hourly intervals for 48 and 96 hrs
respectively produced CL responses in a single individual with a range of 618 to 2946 mV. There was no evidence of periodicity in CL activity over the time periods examined.
Since peak CL responses showed such large variations between individuals, integrated CL values were also measured. Variations between and within individuals similar to those recorded by peak CL were seen in these results.
iii
To examine the possible role of genetic differences in neutrophil function on the variability of CL, pairs of bovine monozygous twins were sampled. There was no correlation in CL response between genetically identical animals with the CL values from pairs of animals differing by as much as 2943 �V.
The effect of cortisol on PMN CL was assessed. Synthetic corticosteroid in vivo and in vitro did not increase the peak CL response from isolated neutrophils.
Profiles produced by recording CL against time were examined. Some cell isolates produced single peaked profiles while others gave a double peaked response. Single and double peaked profiles were recorded from the same donor at different times during a 24 hr period. Storage of the cells for prolonged periods sometimes resulted in an increase in the magnitude of the first peak possibly indicating an increase in the amount of more readily available myeloperoxidase (MPO). Prominent first peaks were still displayed after the cells were washed and resuspended in fresh media suggesting that the more readily available MPO was cell attached rather than truely extracellular.
Neutrophils from ceroid lipfuscinosis-affected sheep produced peak CL responses and CL profiles similar to those given by normal sheep. These results did not confirm the postulated myeloperoxidase deficiency of this condition.
It is concluded that ovine neutrophil CL is subject to large variations which cannot be controlled by standardising the cell isolation and CL analysis techniques. The assay is therefore unsuitable as a measure of neutrophil function where single samples are examined. Where there are consistant differences between individuals over a number of days, then CL may be of use when considered in conjunction with other tests of PMN function.
V
ACKNOWLEDGEMENTS
I wish to express my gratitude to my supervisor, Dr K.M. Moriarty, for his advice, enthusiasm and above all, for his encouragement throughout this study.
I would also like to thank the following people within the Veterinary faculty:
Professor B. W. Manktelow for the original encouragement to undertake postgraduate study.
Dr K.R. Lapwood and Mr J. Elgar for their assistance with the cortisol assays.
Dr R.D. Jolly for providing the CLF-affected sheep and offering advice on the chapter involving these animals.
Mr M. Thomas for assistance in handling the animals.
Mr P. Wildbore for obtaining reagents and coping impatience when these were unavoidably delayed.
with my
Finally, I wish to thank the Phyllis Irene Grey Fellowship for providing the financial assistance without which
could not have been undertaken.
this project
1 .
ABSTRACT
ACKNOWLEDGEMENTS CONTENTS
INDEX OF F IGURES INDEX OF TABLES
THE POLYMORPHONUCLEAR NEUTROPHIL GRANULOCYTE 1 . 1 Origin
1 . 2 Developmen t 1 . 3 Structure 1 . 4 Kinet ics 1. 5 Chemo t axis 1 . 6 Phagocytosis 1 . 7 Degranulation 1 . 8 Respiratory burst 1 . 9 Oxidat ive metabolism 1 . 10 Microbial k illing
a . Oxidat ive bactericidal mechanisms b . Non-oxidative bactericidal mechanisms
1 . 1 1 Neutrophil funct ion tests
a . Nitroblue tetrazolium reduction b . Microbial k illing t ests
c. Chemilumines cence
Page i i
V
vi ix xi 1 1 7 9 1 2 1 6 2 0 2 2 2 3 2 9 3 1 3 4 3 6 3 7 3 7 38 39 2 . THE DETERMINATION OF OPTIMAL CHEMILUMINESCENCE ASSAY
CONDITIONS USING OVINE POLYMORPHONUCLEAR LEUCOCYTES 46
2 . 1 Introduct ion 46
2 . 2 Materials and Methods 4 7
a . Animals
b . Harves t ing o f PMN c . Luminal
d . Latex beads e . Haemoglob in
f . Chemiluminescence analys is 2 . 3 Result s
a . Ef fect of cell concentration
4 7 4 7 4 8 4 8 4 9 4 9 50 50
3 .
vii Page b . Effect o f latex concentrat ion
c . Effect of luminol concentration d . Control s
e . Replication
f . Effect of haemoglobin on CL
g . Effect of s torage o f the isolated cells 2 . 4 Discus sion
VAR IATION IN NEUTROPHIL CHEMILUMINESCENCE ACTIVITY 3 . 1 Introduct ion
5 0 5 0 5 0 5 3 5 3 5 3 5 3 6 1 6 1 6 2 6 2 6 3 6 3 63 3 . 2 Materials and Methods
a. Animals
b . Blood collec t ion c . Harves t ing o f PMN
d . Chemiluminescence analysis
e . Blood l eucocyte counts and plasma cortisol
determinations 64
f . The effect o f corticosteroid in vivo on CL 64 g . The effect of corticos teroid in vitro on CL 6 5
3 . 3 Resul t s 6 5
a . Day-to-day variation i n peak CL values 6 5 b . Day-to-day variat ion i n integrated C L values 6 5 c . Variab ility i n peak CL from samples taken a t
4 and 6 hourly intervals 6 6
d . CL� leucocyte counts and plasma cort isol 66 e. Effect of corticosteroid in vivo 6 9 f . Effect of corticos teroid in vitro 6 9 g . Variations i n neutrophil C L between animals
of identical genet ic composition 3 . 4 Discussion
6 9
4 . PROFILES OF LUMINOL-ENHANCED CHEMILUMINESCENCE RESPONSES 7 7 4 . 1 Introduct ion
4 . 2 Materials and Methods a . Animals
b. Neutrophil isolat ion
c . Chemiluminescence analysis d . Po st-equilibration washing
7 7 7 8 7 8 7 8 7 8 7 8
4 . 3 Results
a . Prof iles of the CL response in normal sheep
b . Prof iles of the CL response in samples
taken at 4 hourly intervals
c . Effect o f incubation a t 2 7°C on the CL pro f ile
d. Effect of pos t-equilibation washing on the CL profile
4.4 Discuss ion
5 . CHEMILUMINESCENCE IN CEROID LIPOFUSCINOSIS-AFFECTED SHEEP
6 .
5 . 1 Introduction
5 . 2 Materials and Methods a . An imals
b . Neutrophil isolation
c . Chemiluminescence analysis 5 . 3 Results
a . Peak CL values
b . Prof iles of the CL responses in ceroid lipo fusc inos is-affected sheep
c . Effect of incubat ion at 2 7°C on the CL pro f ile of ceroid lipofuscinosis-affected sheep
5 . 4 Discuss ion
A CONCLUDING STATEMENT REFERENCES
APPENDIX
Page 7 9 7 9
7 9
8 3
8 3 8 3
9 1 93 93 93 9 3 93 93 93
94
94 96 1 03 1 05 1 5 3
INDEX OF FIGURES
1 . 1 Haematopoietic cell development
1 . 2 Sugges ted intracellular events following phagocytosis 1 . 3 Suggested oxidat ive interactions involved in
respiratory bur s t activity
1 . 4 Possible interrelat ionship of the reactive oxygen species o f the resp iratory burst
1 . 5a Luminol ( 5-amino-2�3-dihydro-1 , 4-phthalazinedione) 1 . 5b Aminophthalate ion
2 . 1 The relat ionship between chemiluminescence and cell concentration
2 . 2 The effect of latex concentration on chemiluminescence 2 . 3 The effect o f luminol concentration on chemiluminescence 2 . 4 The effect o f haemoglobin on luminol-enhanced
chemiluminescence from isolated neutrophils
2 . 5 Mean peak chemiluminescence from isolated neutrophils stored at 4°C
3 . 1 The mean peak chemiluminescence values from 3 sheep over 5 successive days
3 . 2 The mean peak chemilumines cence from 2 sheep bled at 4 hourly intervals for 48 hr
3 . 3 The mean peak chemilumines cence from 2 sheep b led at 6 hourly intervals for 96 hr
3 . 4 Neutrophil chemiluminescence , leucocyte counts and plasma cortisol from a single donor bled via an indwelling cannula at 6 hourly intervals
4 . 1 Profiles o f chemiluminescence responses of neutrophils isolated from 4 dif ferent sheep
4 . 2a Sheep a , chemiluminescence profiles recorded at 4 hourly intervals
ix
Page 3 2 8
3 2
33 42 42
5 1 52 52
5 5
5 6
67
70
7 1
7 3
80
8 1
Page 4 . 2b Sheep b , chemiluminescence profiles recorded at 4
hourly intervals 82
4 . 3a E f fect o f incubation at 2 7°C on the chemiluminescence
profiles from isolated neutrophils (normal sheep) 86 4 . 3b Ef fect of incubation at 2 7°C on the chemiluminescence
profiles from isolated neutrophils (normal sheep) 87 4 . 4 E f fect o f post-equilibrat ion washing on the
chemiluminescence profile
5 . 5 Chemiluminescence profiles o f ceroid lipofuscinos is-
88
a ffec ted lambs 9 7
5 . 2 Chemiluminescence profiles o f normal , Southdown lambs 98 5 . 3a E f fect of incubat ion on the chemiluminescence pro files
of ceroid lipofuscinosis-affected animals 100 5 . 3b E ffect of incubat ion on the chemiluminescence profiles
o f ceroid lipofuscinos is-affected animals 1 0 1
2 . 1
3 . 1
INDEX OF TABLES
Replication of chemiluminescence values within samples
Mean integrated chemiluminescence values from 3 sheep recorded on 4 success ive days
3 . 2 The effect o f corticosteroid in vivo (2mg/kg body weight on mean peak chemiluminescence values
3 . 3 The effect o f corticosteroid in vitro on mean peak chemiluminescence values
3 . 4 Mean peak chemiluminescence responses o f pairs of bovine monozygous twins
4 . 1 Peak chemiluminescence values from the sheep
5 . 1
from which chemiluminescence profiles were recorded at 4 hourly intervals over 24 hr Peak chemiluminescence response s from 7 ceroid lipofuscinos is-affected sheep
xi
Page
54
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68
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74
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