Arch Dermatol Res (1991) 283:362--365
Archives of
9 Springer-Verlag1991
Effect of tumour necrosis factor alpha in vivo on human granulocyte oxidative metabolism A. Kapp, A. Komann, and E. Schi~pf Department of Dermatology, University of Freiburg, Hauptstrasse 7, W-7800 Freiburg, Federal Republic of Germany Received March 2, 1991
Summary. Tumonr necrosis factor alpha (TNF~) effectively stimulates the oxidative metabolism of human P M N in vitro. Moreover, preincnbation of P M N with TNF~ has been shown to result in an altered response of the target cells to subsequent stimulation. In the present study the response of P M N to stimulation in vitro was investigated in patients with metastasizing malignant melanoma receiving bolus injections of recombinant human TNF~ as therapy. TNF~ was given daily for 5 days. Blood samples were taken prior to TNF~ administration on days 1 to 4 and on day 8. Lucigenin-enhanced chemiluminescence (CL) was used as a sensitive measure of granulocyte oxidative metabolism. P M N were stimulated with TNF~, TNF~, GM-CSF, P M A , opsonized zymosan and f-met-leu-phe. A significant increase in CL responses was detected upon stimulation with TNF~, TNF~ and PMA from day 1 to day 3, whereas no significant changes were observed for the background activity or when GM-CSF or opsonized zymosan were used as stimuli. On day 4 all CL responses returned to the day 1 starting level. A further significant decrease was observed on day 8 upon stimulation with TNF~, TNF~ and GMCSF. In contrast, the effect induced by f-met-leu-phe reached a maximum on day 4, but the CL response was found to be at the starting level on day 8. The results indicate that TNF~ induces significant changes in P M N response to distinct stimuli in vivo. Moreover, it may be possible that continous daily infusions with TNF~ induce a hyposensitization of PMN oxidative metabolism.
the most important granulocyte-activating mediator with respect to its capacity to stimulate the release of toxic reactive oxygen species [8, 10]. Previously, it has been shown that preincubation of granulocytes with T N F a in vitro significantly modulates the respons e of these cells to subsequent stimulation with different cytokines and stimuli of the oxidative burst [8]. A significant modulation of h u m a n granulocyte function has been reported in patients receiving therapy with G M - C S F [3, 14] or G - C S F [12]. In the present study, granulocyte oxidative metabolism was investigated ex vivo in patients with metastasizing malignant melanoma undergoing therapy with bolus injections of recombinant h u m a n TNF~.
Materials and methods
Design of the study Four patients with metastasizing malignant melanoma were investigated. Within the scope of a clinical study these patients received daily intravenous infusions of recombinant human TNF~ for 5 days. TNF~ was given as a bolus infusion at a concentration of 400 ~tg (specific activity 8.5 • 1 0 6 U/mg) in 100 ml saline, containing 5 mg/ ml human serum albumin (HSA). A concentration of 400 p.g TNFc~/ 6 1blood corresponds to a putative concentration of 600 U/ml. Blood samples for measurement of granulocyte oxidative metabolism ex vivo were taken 15 rain prior to administration of TNF~ on day 1, 2, 3 and 4, and, as a control, on day 8.
Key words: T N F a - P M N - Oxidative metabolism Chemiluminescence
Isolation of polymorphonuclear neutrophilic granuIocytes
Only selected cytokines are capable of directly activating the oxidative metabolism of h u m a n polymorphonuclear neutrophilic granulocytes (PMN) [4]. Besides of G M CSF and tumour necrosis factor beta (TNFfl), T N F a is
Offprint requests to: A. Kapp
PMN were isolated from heparin-anticoagulated venous blood of the patients as previously described [6, 7] by rico11 gradient centrifugation and three 30 s cycles of 0.2% NaC1 treatment followed by the addition of an equal volume of 1.6% NaC1 to lyse red blood cells. Cells were more than 95 % viable as estimated by trypan blue exclusion. Subsequently PMN were suspended to a density of 5 • 1 0 6 cells/ml in HEPES-buffered Hank's balanced salt solution (HBSS), pH 7.4, containing 2 mM lucigenin and 1 mg/ml bovine serum albumin (BSA).
A. Kapp et al.: In vivo-granulocyte activation by TNF~ patients
363 patients
xl~ 8
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1
300'
1 /
1 /
/
/
1
800.
1
1
1
TNF
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Fig. 1. CL response upon stimulation in vitro of isolated PMN from patients during therapy with TNFe. TNFc~ ( 0 - . - 0 ) , TNF/? ( 0 . . . . 0), and GM-CSF ( 0 - - 0 ) were used as stimuli. 9 - - - 0 , background activity induced by addition of medium. Counts integrated over 60rain were recorded. * p_
Archives of
9 Springer-Verlag1991
Effect of tumour necrosis factor alpha in vivo on human granulocyte oxidative metabolism A. Kapp, A. Komann, and E. Schi~pf Department of Dermatology, University of Freiburg, Hauptstrasse 7, W-7800 Freiburg, Federal Republic of Germany Received March 2, 1991
Summary. Tumonr necrosis factor alpha (TNF~) effectively stimulates the oxidative metabolism of human P M N in vitro. Moreover, preincnbation of P M N with TNF~ has been shown to result in an altered response of the target cells to subsequent stimulation. In the present study the response of P M N to stimulation in vitro was investigated in patients with metastasizing malignant melanoma receiving bolus injections of recombinant human TNF~ as therapy. TNF~ was given daily for 5 days. Blood samples were taken prior to TNF~ administration on days 1 to 4 and on day 8. Lucigenin-enhanced chemiluminescence (CL) was used as a sensitive measure of granulocyte oxidative metabolism. P M N were stimulated with TNF~, TNF~, GM-CSF, P M A , opsonized zymosan and f-met-leu-phe. A significant increase in CL responses was detected upon stimulation with TNF~, TNF~ and PMA from day 1 to day 3, whereas no significant changes were observed for the background activity or when GM-CSF or opsonized zymosan were used as stimuli. On day 4 all CL responses returned to the day 1 starting level. A further significant decrease was observed on day 8 upon stimulation with TNF~, TNF~ and GMCSF. In contrast, the effect induced by f-met-leu-phe reached a maximum on day 4, but the CL response was found to be at the starting level on day 8. The results indicate that TNF~ induces significant changes in P M N response to distinct stimuli in vivo. Moreover, it may be possible that continous daily infusions with TNF~ induce a hyposensitization of PMN oxidative metabolism.
the most important granulocyte-activating mediator with respect to its capacity to stimulate the release of toxic reactive oxygen species [8, 10]. Previously, it has been shown that preincubation of granulocytes with T N F a in vitro significantly modulates the respons e of these cells to subsequent stimulation with different cytokines and stimuli of the oxidative burst [8]. A significant modulation of h u m a n granulocyte function has been reported in patients receiving therapy with G M - C S F [3, 14] or G - C S F [12]. In the present study, granulocyte oxidative metabolism was investigated ex vivo in patients with metastasizing malignant melanoma undergoing therapy with bolus injections of recombinant h u m a n TNF~.
Materials and methods
Design of the study Four patients with metastasizing malignant melanoma were investigated. Within the scope of a clinical study these patients received daily intravenous infusions of recombinant human TNF~ for 5 days. TNF~ was given as a bolus infusion at a concentration of 400 ~tg (specific activity 8.5 • 1 0 6 U/mg) in 100 ml saline, containing 5 mg/ ml human serum albumin (HSA). A concentration of 400 p.g TNFc~/ 6 1blood corresponds to a putative concentration of 600 U/ml. Blood samples for measurement of granulocyte oxidative metabolism ex vivo were taken 15 rain prior to administration of TNF~ on day 1, 2, 3 and 4, and, as a control, on day 8.
Key words: T N F a - P M N - Oxidative metabolism Chemiluminescence
Isolation of polymorphonuclear neutrophilic granuIocytes
Only selected cytokines are capable of directly activating the oxidative metabolism of h u m a n polymorphonuclear neutrophilic granulocytes (PMN) [4]. Besides of G M CSF and tumour necrosis factor beta (TNFfl), T N F a is
Offprint requests to: A. Kapp
PMN were isolated from heparin-anticoagulated venous blood of the patients as previously described [6, 7] by rico11 gradient centrifugation and three 30 s cycles of 0.2% NaC1 treatment followed by the addition of an equal volume of 1.6% NaC1 to lyse red blood cells. Cells were more than 95 % viable as estimated by trypan blue exclusion. Subsequently PMN were suspended to a density of 5 • 1 0 6 cells/ml in HEPES-buffered Hank's balanced salt solution (HBSS), pH 7.4, containing 2 mM lucigenin and 1 mg/ml bovine serum albumin (BSA).
A. Kapp et al.: In vivo-granulocyte activation by TNF~ patients
363 patients
xl~ 8
xlO-6
1
300'
1 /
1 /
/
/
1
800.
1
1
1
TNF
,
\
,// 200'
1 ]'NF
6O0
\
/
\
g
\
U
2
400
I/'/
0 0
I .o'" .
\\\
100'
•
~
,/
200
/
"'.~--~
T'f--'-'"
I
,
,
1
2
3 doy
L
"I II
,
il- .......
~ ....
,
,
8
i
2
3
~
Fig. 1. CL response upon stimulation in vitro of isolated PMN from patients during therapy with TNFe. TNFc~ ( 0 - . - 0 ) , TNF/? ( 0 . . . . 0), and GM-CSF ( 0 - - 0 ) were used as stimuli. 9 - - - 0 , background activity induced by addition of medium. Counts integrated over 60rain were recorded. * p_
