Tumor Necrosis Factor Primes Neutrophils by Shortening the Lag Period of the Respiratory Burst

Tumor Necrosis Factor Primes Neutrophils by Shortening the Lag Period of the Respiratory Burst JAMES R. HUMBERT, MO,

ELSA L. WINSOR, PHD

ABSTRACT: Pretreatment of neutrophils (PMNs) with low-dose tumor necrosis factor (TNF) enhances their capacity to produce oxidant radicals after stimulation with a variety of agents ('priming'). We used a continuous cytochrome C assay to investigate the superoxide production by human PMNs primed with TNF and subsequently stimulated with phorbol myristate acetate (PMA). There was no difference in the maximum rate of superoxide production by primed and unprimed PMNs stimulated with either high (2 X 10-6 M) or low levels of PMA (2 X 10-8 M). Following stimulation with high levels of ~MA, primed PMNs demonstrated a significantly shorter lag period than unprimed cells (103.3 ± 14.4 vs. 142.1 ± 21.7 seconds) and larger amounts of superoxide generated in the intervals between 100 seconds (3.1 ± 0.5 vs. 1.7 ± 0.3 nmol/106 PMNs) and 300 seconds (14.9 ± 1.2 vs. 12.2 ± 1.1). Primed cells stimulated with low levels of PMA (2 X 10-8 M) displayed a significantly shorter lag period (1225.8 ± 96.8 vs. 1573.8 ± 74.3 seconds) and a greater production of superoxide between 1300 seconds (5.4 ± 0.9 vs. 3.0 ± 0.4 nmol/10 6 PMNs) and 1900 seconds (25.7 ± 4.3 vs. 14.9 ± 2.4) than unprimed cells. These results indicate that priming of PMN s with TNF increases superoxide production during the early phase of the respiratory burst through a shortening of the post-stimulation lag period. KEY INDEXING TERMS: Neutrophil; Priming; Respiratory Burst; Superoxide; Tumor N ecrosis Factor. [Am J Med Sci 1990; 300(4): 209-213.]

From the Department of Pediatrics, Tulane University Medical School, New Orleans, Louisiana. This work was presented in part at the 1989 meeting of the Southern Society for Pediatric Research in New Orleans, LA. Correspondence: James R. Humbert, MD, Department of Pediatrics, Tulane University Medical School, 1430 Tulane Ave, New Orleans, LA 70112.

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n high doses, tumor necrosis f~ctor (TNF) dire.ctly stimulates several neutrophil (PMN) functions (phagocytosis, 1 respiratory burst, hydrogen peroxide production,2 chemoluminescence,3 and adherence4 ) while it inhibits the migration of these cells. 3 Low doses of TNF do not directly stimulate PMNs but modify ("prime") these cells so that they respond more vigorously to a subsequent stimulus; superoxide production following stimulation with phorbol myristate acetate (PMA) or n-formyl-methionyl-leucylphenylalanine (FMLP) is markedly enhanced after a short incubation of PMNs with low-dose TNF.5 The exact molecular basis for the TNF -induced priming has not been defined; furthermore, the kinetics of priming have received little attention because most investigators have worked with discontinuous assays of superoxide production. In this article, we investigated the kinetics of superoxide production by PMNs that were primed with TNF and stimulated with PMA. Our findings suggest that the previously reported increase of superoxide production by TNFprimed PMNs is due primarily to a significant shortening of the lag period between the stimulation of the cells with PMA and their subsequent respiratory burst.

I

Methods

PMNs, prepared from blood collected from healthy adult volunteers and anticoagulated with preservative-free heparin (10 Vlml), were separated using a two phase gradient of methyl cellulose/hypaque and Ficoll-hypaque6 followed by lysis of residual erythrocytes with 0.87% ammonium chloride. The remaining cells were washed once in Hanks balanced salt solution (HBSS) without phenol red (pH 7.2) and suspended to a concentration of 2 X 106 leukocytes per milliliter. The percentage of PMNs was determined by routine differential examination and varied between 94% and 99%. Tumor necrosis factor was generously provided by the Cetus Corporation (Emeryville, CA) as a lyophilized preparation with a concentration of 24 X 106 V I mg. The material was reconstituted with. water to .a concentration of6 X 106 Vim 1 and frozen In 20 ILl ahquots at -70°C. Prior to use, a working stock of 1

209

Neutrophil Priming by TNF

20

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!::

Rate

10

i5 E c 5

0.~6=='~4=='~2~~~~4~~6--~8--~10--1~2--1~4--1~6--1~8~20 Sec x 100 LAGS

Figure 1. Superoxide production in a typical experiment of tumor necrosis factor (TNF)'priming in neutrophils (PMNs) stimulated with high·dose phorbol myristate acetate (PMA) (2 X 10- 6 M). The only difference between the TNF-primed sample and the unprimed (control) sample is the earlier start of the respiratory burst (the shorter lag period). o = Unprimed, PMA. 0 = Primed, PMA.• = Primed, no PMA.

X 106 U Iml was prepared. In priming experiments the final concentration of TNF varied from 10 U Iml to 5000 U ImL The manufacturer determined that the endotoxin level of TNF was less than 0.02 ng/mL Superoxide production was measured using a continuous cytochrome C reduction assay7 conducted in a Beckman DU62 spectrophotometer fitted with a Peltier attachment to maintain the cells at 37°C. Each cuvette contained 1.0 ml with 1.8 X 106 leukocytes and 0.625 mg of Cytochrome C (Sigma, St. Louis, MO). Superoxide dismutase (SOD) was added to a duplicate of each cuvette in a final concentration of 20 ug/mL The mixtures equilibrated for 10 minutes at 37°C in the spectrophotometer, and TNF or HBSS was added; the cuvettes were mixed by inversion and maintained at 37°C for 10 additional minutes. Phorbol myristate acetate (PMA) was added to a final concentration of 2 X 10-6 M (high dose) or 2 X 10-8 M (low dose); the cuvettes were mixed by inversion and the absorbance at 550 nm recorded every 20 seconds for 30 to 60 minutes. The nmol superoxide/l06 PMNs produced was calculated using the extinction coefficient of 21 X 103 cm21M. 7 Lag period was defined as the interval between stimulation by PMA and the time of onset of the respiratory burst, as determined by extrapolating the line formed during the maximum rate of superoxide production to the base line. Maximum rate of superoxide production was calculated from the steepest linear section of the respiratory burst (Fig. 1). Data were analyzed using the two-tailed paired t test andlor Student's t test as appropriate.

210

Results

A typical experiment with high-dose PMA is shown in figure 1. Primed and unprimed cells stimulated with PMA yielded similar production rates of superoxide. However, the onset of the respiratory burst started earlier in TNF -primed cells than in unprimed cells. Without PMA stimulation, TNF -primed PMN s generated levels of superoxide that were marginally above baseline. TNF significantly shortened the lag period (i.e., the time between the addition of PMA and the onset of the respiratory burst). With high-dose PMA, the lag period was shortened in primed PMNs from 142 seconds to 103 (p < 0.05); washing cells did not abolish this shortening of the lag period (data not shown). When we used low-dose PMA, we again observed a significant shortening of the lag period in primed samples, but samples treated with low-dose PMA displayed a lag period ten-times longer than PMNs stimulated with high dose PMA (Table 1, Fig. 2). TNF priming did not affect the maximum rate of superoxide production in our experiments. Low-dose PMA did produce a slightly higher maximum rate of superoxide production 'in primed than in unprimed samples, but this difference was not statistically significant. With both primed and unprimed samples, high-dose PMA yielded rates of superoxide production about four times greater than low-dose PMA. The amount of superoxide produced at selected post-stimulation intervals was significantly different between primed and unprimed cells for samples stimulated with high dose PMA during the period from 100 to 300 seconds, after which the differences were not significant (Table 3). Our examination of the groups of individual pairs (not shown) and of their composite graph (Fig. 2) demonstrated that the increase in superoxide production between 100 and 300 seconds in TNF-primed samples was due to an earlier start of the respiratory burst (shorter lag period). Samples stimulated with low-dose PMA also showed significantly higher amounts of superoxide produced Table 1. Shortening of the Lag Period of the Respiratory Burst by Tumor Necrosis Factor (TNF)-Priming at Two Concentrations of Phorbol Myristate Acetate (PMA) Low Dose (n Control TNF-Primed

= 5)

1573.8 ± 74.3 1225.8 ± 96.8 P <0.05

High Dose (n

=

22)

142.1 ± 21.7* 103.3 ± 14.4 * p <0.01

Priming with TNF results in a significantly shorter lag period between the time of PMA stimulation and the onset of the respiratory burst. High dose PMA produces a lag period about 10 times shorter than low dose PMA. Values = seconds (x ± SE) Low dose = 2 X 10-8 M PMA; high dose = 2 X 10-6 M PMA. * p < 0.05 when compared with low dose values.

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Humbert and Winsor

A 30

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40

Sec X 100 Figure 2. Superoxide produced by TNF-primed and unprimed neutrophils (PMNs) stimulated with high-dose (2 X 10-6 M) or low dose (2 X 10-8 M) phorbol myristate acetate (PMA). A. High dose (2 X 10-6 M) PMA; results represent the mean of 22 paired experiments. B. Low-dose (2 X 10- 8 M) PMA; results represent the mean of five paired experiments. 0"" Unprimed. 0 "" Primed.

in TNF -primed samples, again because of a shortened lag period, but these apparent increases in superoxide occurred in the intervals from 1300 to 2500 seconds after PMA stimulation. On the other hand, the conditions of our experiments were such that all of the cytochrome present in the assay was reduced, thus precluding any conclusions about a difference, or lack thereof, in the maximum amount of superoxide produced between primed and unprimed samples (Table 2, Fig. 2). Discussion

Several authors have examined the oxidative burst of TNF-primed human PMNs in response to soluble or particulate stimuli and generally have concurred

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that the burst is enhanced by priming. 1- 3 ,5 An apparent increase in superoxide production by TNFprimed cells over unprimed cells has been reported by most investigators who used single measurements of superoxide at arbitrary intervals after the application of a stimulus. For instance Berkow et al,5 using a discontinuous assay of superoxide production, demonstrated a significant increase in superoxide production five minutes after stimulation with PMA (concentration: 2.5 X 1O-8 M) in PMN s primed with 1000 U TNFIml; this enhancement approached 150% of control (non-primed) values. Using a similar low-dose PMA (2 X 10-8 M), we found no such enhancement at five minutes following stimulation but measured clearly higher superoxide values during the interval from 1300 to 2500 seconds after stimulation (Fig. 2, Table 3). This discrepancy seems due to the long lag period we observed in our low-dose PMA experiments; once the respiratory burst began, we found large differences in superoxide production between primed and unprimed PMNs that occurred at intervals between 1300 and 2500 seconds after stimulation and were due mostly to the earlier start (shorter lag) of the respiratory burst of primed PMNs. The maximal rate of superoxide production was not significantly increased in our primed samples (Table 2). With high dose PMA, the shortening of the lag period accounted entirely for the increase in superoxide production of primed PMNs during a post-stimulation period of 100 to 300 seconds (Fig. 2, Table 3). Ferrante et al3 "primed" human PMNs with TNF (100 U/5 X 105 cells) and, at 30 minutes after stimulation with various PMA concentrations, measured an increase in hydrogen peroxide production. That point (1800 seconds) corresponds to one of the intervals in which we observed a large increase in oxidative activity by TNF-primed PMNs after low-dose PMA stimulation. Thirty minutes after stimulation with highdose PMA, these same authors observed much higher hydrogen peroxide levels than those obtained with low-dose PMA but measured no difference between primed and unprimed samples. This pattern of oxidative response agrees with our observations (Fig. 2): the lack of difference in oxidative activity at 30 minutes between primed and unprimed PMN s stimulated with high dose PMA is due to the much earlier onset of the respiratory burst and possibly also its earlier completion, although our experiments were not designed to determine the latter. Using a flow cytometry-based measurement of dichlorofluorescein diacetate, Livingston et al8 reported a significant increase in respiratory burst activity following stimulation of TNF -primed human PMNs with Staphylococcus aureus. That activity was greater than a 100% increase above control values when 10 Ulml of TNF was used as a priming agent and was measured five minutes and 15 minutes after 211

Neutrophil Priming by TNF

Table 2. Effect of Two (PMA) Concentrations on the Respiratory Burst Low Dose (n Unprimed Maximum rate superoxide production (nmol/106 PMNs/min)

1.2 ± 0.05

= 5)

High Dose (n

TNF-Primed

Unprimed

1.4 ± 0.1

5.0 ± 0.4*

27.6 ± 5.1

29.7 ± 5.4 p= ns

TNF-Primed 5.3 ± 0.3* p = ns

p= ns

Maximum amount superoxide production (nmol/106 PMNs)

= 22)

24.7 ± 0.9

24.9 ± 0.9 p = ns

TNF priming of PMNs does not affect the rate of superoxide production or the maximum amount of superoxide produced (the latter being due to complete reduction of all cytochrome C in our assays). High-dose PMA results in a significantly higher rate of superoxide production, and a shorter respiratory burst, than low-dose PMA. Numbers in parenthesis = pairs of experiments. Results = x ± SE. * p < 0.05 when comparing high dose with corresponding low dose values; ns = not significant.

adding the bacterial stimulus. Although these authors reported no formal measurements of oxidative rate, our examination of their data suggests similar rates of oxidative activity over the 15 minute reaction time for primed and unprimed samples. Particulate (bacterial) stimulation produces an intense oxidative burst reaction from PMNs similar to that observed after high dose PMA stimulation. Thus, these authors' results correlate well with our findings of a significantly increased amount of superoxide in TNF -primed PMNs 300 seconds (5 minutes) after high dose PMA stimulation, even though these cells demonstrated the same maximal rate of superoxide production as unprimed controls (Tables 2 and 3, Fig. 2). Berkow and Dodson,9 using a continuous assay of superoxide measurement, concluded that priming-induced enhancement of superoxide production was acTable 3. Superoxide Production by Tumor Necrosis Factor (TNF)-Primed Neutrophils (PMNs) at Various Intervals after Phorbol Myristate Acetate (PMA) Stimulation Low Dose PMA (n = 5)

100 sec 200 sec aoo sec 1aOO sec 1900 sec 2500 sec

High Dose PMA (n = 22)

Control

TNF-Primed

Control

TNF-Primed

0.8 ±0.5 0.8 ±0.5 0.9 ± 0.5 3.0 ±0.4 6.8 ± 1.5 14.9 ± 2.4

1.0 ± 0.4 1.1 ± 0.4 1.1 ±0.4 5.4 ± 0.9* 14.2 ± 2.a* 25.7 ±4.a*

1.7 ± 0.3 6.0 ± 1.0 12.2 ± 1.4 nd nd nd

3.1 ±0.5* 8.1 ± 1.1* 14.9 ± 1.2* nd nd nd

Low-dose PMA produces a significantly higher amount of superoxide between 1300 and 2500 seconds after stimulation in TNF-primed PMNs. In TNF-primed PMNs stimulated with high-dose PMA, this increase is observed earlier, between 100 and 300 seconds after stimulation. All values = nmol/IOS PMNs. Results = X ± SE. * p < 0.05, comparing primed with unprimed samples. p < 0.05, comparing high-dose with low-dose PMA values. Low dose PMA = 2 X 10-8 M. High dose PMA = 2 X 10-6 M. nd = not done.

212

companied both by a shorter lag period and by an increase in the initial (maximal) rate of production of superoxide, when PMA doses of 2 X 10-9 M to 2.5 X 10-6 M were used as stimulant. These results agree with our observations of the shortening of the lag period; however, they are at variance with our inability to demonstrate a significant increase in maximal rate of superoxide production, possibly due to differences in experimental conditions. Tennenberg and Solomkinlo found no increase in maximal rate of superoxide production in their TNF-primed PMNs after PMA stimulation in a continuous assay system similar to ours but did observe an increase in rate following FMLP stimulation; they made no comment regarding the effect of TNF priming with either stimulus on the lag period. Our findings, and the bulk of the data from the literature, can thus be tentatively summarized as follows: priming ofPMNs with TNF renders these leukocytes extremely susceptible to PMA stimulation at any dose; such stimulation results, through a shortening of the lag period, in an earlier onset of the respiratory burst. This, rather than an increase in the maximal rate of superoxide production, accounts for the increases in superoxide amounts observed at certain post-stimulation times, especially when a relatively high-dose PMA is used as the stimulant of the respiratory burst. The possible molecular background for the shorter lag period secondary to TNF priming (the creation in effect, of 'hair-trigger' PMNs) is unclear at present. Our results and those of Berkow et al5 and Tennenberg and Solomkin lo reveal that the TNF-priming effect cannot be removed by washing the cells, thus suggesting an irreversible change in the cellular machinery concerned with the onset of the oxidative burst. TNF-induced changes in the kinetics of the NADPH-oxidase enzyme responsible for the oxidative burst are unlikely9 since activation of that en-

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Humbert and Winsor

zyme seems to be an all-or-nothing event.l1 Because PMA acts directly on the cytosolic C-kinase that inaugurates the intracellular cascade of oxidation reactions,12 a change in behavior of that enzyme was suspected but not found. 9 If TNF priming promotes degranulation, then translocation of some of the oxidative machinery from granule components to the cell membrane could account in part for the shortened lag period; however, priming doses of TNF have not been shown to enhance degranulation in response to PMA, although it does so in response to FMLP.lO Similarly, while increased expression 10 or affinity 13 of FMLP receptors has been demonstrated following TNF priming, no such enhancement has been documented to date for PMA receptors. The unexplored territory at present includes the cooperative activity of the several different cytosolic proteins that together seem responsible for assembling the different components of the NADPH-oxidase. 14 TNF priming does increase phosphorylation of as yet unidentified PMN proteins;9 whether priming involves a conformational, phosphorylation-dependent change of one or several of the cytosolic proteins is conjectural at present and certainly deserves further study. References 1. Shalaby MR, Aggarwal BB, Rinderknecht E, Svedersky LP, Finkle BS, Palladino MA, Jr: Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors. J Immunol135:2069-2073, 1985. 2. KlebanoffSJ, Vadas MA, Harlan JM, Sparks LH, Gamble JR, Agosti JM, Waltersdorph AM: Stimulation of neutrophils by tumor necrosis factor. J Immunol136:4220-4225, 1986. 3. Ferrante A, Nandoskar M, Walz A, Goh DHB, Kowanko IC: Effects of tumor necrosis factor alpha and interleukin-1 alpha and beta on human neutrophil migration, respiratory burst and degranulation. Int Arch Allergy Appl Immunol 86:82-91, 1988.

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4. Gamble JR, Harlan JM, Klebanoff SJ, Vadas, MA: Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Med USA 82:8667-8671, 1985. 5. Berkow RL, Wang D, Larrick JW, Dodson RW, Howard TH: Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. J Immunol139:3783-3791,1987. 6. Boyum A: Isolation of leucocytes from human blood. A twophase system for the removal of red cells with methylcellulose as erythrocyte-aggregating agent. Scan J Clin Lab Invest 21 (Suppl. 97):9-29, 1968. 7. Cohen RJ, Chovaniec ME. Superoxide generation by digitonin-stimulated guinea pig granulocytes: a basis for a continuous assay for monitoring superoxide production and for the study of the activation of the generating system. J Clin Invest 61: 1081-1087,1978. 8. Livingston DR, Appel SH, Sonnefeld G, Malangoni MA: The effect of tumor necrosis factor and interferon on neutrophil function. J Surg Res 46:322-326, 1989. 9. Berkow RL, Dodson MR: Biochemical mechanisms involved in the priming of neutrophils by tumor necrosis factor. J Leukocyte Biol44:345-352, 1988. 10. Tennenberg SD, Solomkin JS: Activation ofneutrophils by cachectin/tumor necrosis factor: Priming of N-formyl-methionyl-Ieucyl-phenylalanine-induced oxidative responsiveness via receptor mobilization without degranulation. J Leukocyte Biol47:217-223,1990. 11. Bellavite P: The superoxide-forming enzymatic system of phagocytes. Free Radical Biol Med 4:225-261, 1988. 12. Smith RJ, Justen JM, Sam LM: Function and stimulus-specific effects ofphorbol12-myristate 13-acetate on human polymorphonuclear neutrophils: autoregulatory role for protein kinase C in signal transduction. Inflammation 12:597-611, 1988. 13. Atkinson YR, Marasco WA, Lopez AF, Vadas MA: Recombinant human tumor necrosis factor-alpha: regulation of N-formylmethionylleucylphenylalanine receptor affinity and function on human neutrophils. J Clin Invest 81:759-765, 1988. 14. Curnutte JT, Scott PJ, Mayo LA: The cytosolic components of the respiratory burst oxidase: resolution of four components, two of which are missing in complementing types of gammainterferon-responsive chronic granulomatous disease. Blood 72 (suppl1):144, 1988.

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