CELL BIOCHEMISTRY AND FUNCTION
VOL.
8: 147-155 (1990)
Influence of 4-Hydroxynonenal on Chemiluminescence Production by Unstimulated and Opsonized ZymosanStimulated Human Neutrophils C. DI MAURO, G. CAVALLIT, M. C. AMPRIMOT, L. PARADISI, G. SCANOI, M. CURZIO AND M. U. DIANZANI Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy t Laboratorio di Chimica Clinica, Nuova Astanteria Martini, Piazza Donatori di Sangue, 10125 Torino, Italy
The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, Net-Leu-Phe ( N L P ) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between N L P and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different. KEY
wO~1)~--4-Hydroxynonenal; chemiluminescence; zymosan; neutrophils; respiratory burst.
INTRODUCTION 4-Hydroxy-2, 3-trans-nonenal (HNE) is one of the major products of peroxidative breakdown of arachidonic, y-linolenic agd linoleic acids.' It has been found to be formed during inflammatory and to possess a wide spectrum of biological effects on different cell types and rnolecules.'s4 In particular, in uitro studies showed that HNE in the micromolar range stimulates, in the presence of albumin, the morphological polarization and the oriented migration5 of rat polymorphonuclear neutrophils (PMN). In the case of human PMN it provokes a weak but significant stimulation of the random migration.6 In contrast, M HNE in uitro inhibits PMN-stimulated O i production,' neutrophil migration' and monocyte immune phagocytosis.* Considering the role
played by this aldehyde in phagocyte functions, the aim of the present study was to investigate its influence on the respiratory burst of unstimulated as well as of opsonized zymosan-stimulated human PMN, by means of the chemiluminescence technique in a luminol-amplified system. It is well known that when neutrophil plasma membranes interact with soluble or particulate stimuli, a complex series of events, resulting in a burst of oxidative metabolism associated with an increased glucose oxidation via the hexose monophosphate shunt takes place.' This process includes the generation of O;, other reactive oxygen radicals and H,O, which are produced by NADPH-oxidase and myeloperoxidase systems and that can be detected as light emission (chemiluminescence, CL). ' O - 1 1
Addressee for correspondence: Dr Clelia Di Mauro, Dipartirnento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy. 0203 - 6484/90/030147-09%0.5.00 c1990 by John Wiley & Sons, Ltd.
148 MATERIALS AND METHODS Celt Preparation
Human polymorphonuclear leukocytes were harvested from heparinized peripheral blood of normal adult donors and separated by a modified Boyum's method.' Blood was mixed with 3 per cent dextran in 0-9 per cent NaCl solution. The red cells were allowed to settle for 60min at room temperature. The leukocyte-rich plasma obtained was removed and layered over a Lymphoprep gradient and centrifuged for 18 rnin at 800g to separate PMN. Residual red cells were removed by osmotic shock. PMN were washed twice in Hanks' balanced salt solution without phenol red (HBSS) and adjusted to 5 x lo6 ml-' in HBSS plus 0.4 per cent bovine serum albumin (BSA), whereas cells for phagocyte assays were diluted in buffer plus 0.2 per cent BSA. PMN were always more than 95 per cent of the leukocyte population. Cell viability was always over 94 per cent as judged by the Trypan blue exclusion test and it was not influenced by HNE exposure. In parallel malonaldehyde (MDA) levels were evaluated in blood samples, by the thiobarbituric acid test,I3 in order to investigate peroxidation reactions that might take place during the time of red-cell sedimentation. If so, lipid peroxidation end-products and in particular 4-hydroxynonenal, could influence the CL tests. Results obtained showed that a low level of MDA was produced. Values obtained were in accordance with the observations of Poli et aZ.14 who also demonstrated that 4-hydroxynonenal production was not detectable in red cells from normal subjects.
C DI MAURO E T A L
coupled to a computer (Olivetti M24) with a specific PHAGO-TEST program (LKB) for data integration. Results were represented on graphs with time units on abscissa and light intensity units (mV) on ordinate. The values in millivolts (mV) of peak height were evaluated and are reported in the results. The standard reaction mixture in the cuM Iuminol, vettes consisted of 600 p1 of 2 x 150 p1 of HBSS + 0-4 per cent BSA, 100 pI of cell suspension ( 5 x lo6 PMN ml-I). Cells were always preincubated for 5 min at 37 "C, then 50 pl of buffer (control) or HNE or Net-Leu-Phe (fMLP) were added and chemiluminescence was measured for 20 min. In other experiments cells were incubated for different times (0, 10, 20min) with the abovementioned substances and then they were stimulated by 50pl of serum opsonized zymosan; light emission was detected for 20 min. In addition, to evaluate the effects of SH-compounds in our experimental system, some assays were performed in the presence of cysteine and/or M HNE before zymosan stimulation. Other variations are indicated in the text and legends. Background emission (-0.5 mV) was recorded using samples containing reagents alone, without cells. The CL of resting PMN was obtained by measuring the light emitted by control cells without added stimuli. 4-Hydroxynonenal was a gift from Professor H. Esterbauer (University of Graz, Austria) and was prepared as previously described.s*l 6,1 Luminol and N L P were dissolved in dimethyl sulphoxide (DMSO) at a concentration of loe3 M. All compounds, before use, were diluted in HBSS to the desired concentration.
'
Zymosan Opsonization Zymosan opsonization was performed as previously described" by incubating at 37 "C, for 30 min, 80 mg of zymosan, 2 ml of human pooled AB serum and 2 mI of HBSS. The suspension was centrifuged at 800g for 10min. The pellet was washed twice with 0.9 per cent NaCl and resuspended with 4 ml of HBSS to a final concentration of 20 mg of zymosan m1-I.
Phagocytosis Assay
Samples containing 100 p1 of cell suspension were prewarmed at 37 "C for 5 min before adding 100 p1 of HNE (final concentration in the samples of or lo-' M) or 100p1 of buffer (control). Incubation with the aldehyde was carried out for 20 min, then 50 pl of opsonized zymosan (25,000 particles (mm3)- ') were pipetted into the samples. The reaction was allowed to proceed for 10min and then stopped by placing the tubes in ice. Slides Chemiluminescence Assay with cell suspensions were prepared and stained Measurements of chemiluminescence were per- with May Grunwald-Giemsa. Results were exformed with a luminometer (LKB Wallac 1251) pressed as percentage of phagocytosis (number of
CHYDROXYNONENAL AND HUMAN NEUTROPHIL CHEMILUMINESCENCE
phagocytosing cells (total number of cells)-' x loo). Chemicals
BSA, DMSO, fMLP, Luminol, Trypan blue and Zymosan were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Dextran (p.m. 500 OOO) from Pharmacia, Uppsala, Sweden. Lymphoprep from Nyegard & Co. AS, Oslo, Norway. HBSS from Gibco Grand island, N.Y. Cysteine, malonaldehyde, thiobarbituric acid, were obtained from Merck Darmstadt, West Germany. RESULTS Chemiluminescence Assay Injuence of H N E and f M L P on C L Production by Unstimulated Neutrophils. HNE was tested in a wide range of doses (10-4-10-9 M); none of them was found able to enhance CL production by neutrophils (Figure lA, B). In some assays, CL measurements were prolonged up to 60 min to exclude the presence of a delayed peak, but in no case was an increase in CL detected. On the contrary, and lo-' M HNE decreased CL emission nearly to background values (Figure IA). In parallel, experiments were done by using, as a positive control, PMN in the presence of the soluble tripeptide fMLP at lo-' M. As shown in Figure 2 this peptide greatly increased PMN CL production, a peak of light emission being reached within 2 min. InJEuence of H N E and f M L P on C L Production by Opsonized Zymosan-Stimulated Neutrophils. The effect of HNE on zymosan-stimulated cells was evaluated by incubating samples at 37 "C for 0, 10, 20 min with 10-4-10-9 M HNE before adding zymosan (Figure 3). As it can be seen, and IO-' MHNE exerted a dose-related inhibition of CL production, maximal inhibition values being reached after 20min of incubation; lower HNE concentrations did not significantly modify detectable CL emission. in Figure 4A, B the time course of a typical experiment is shown. Zymosan stimulation of PMN resulted in a rapid increase in light emission reaching a peak within 10 min. HNE did not alter the time to reach the maximum. Cell stimulation with fMLP and zymosan added simultaneously, as
149
well as zymosan addition to cells previously incubated with the tripeptide for 20 min, resulted both in a shortening in the peak time and in an enhancement of CL produced with respect to cells stimulated by zymosan alone (Figure 5). Finally it should be noted that the extent of CL production varied greatly in both fh4LP or zymosan-treated PMN depending on the cell preparation tested. Injuence of Cysteine. The effect of thiols expected to act in uivo by detoxication of reactive aldehydes was also studied. The cells, before being stimulated by opsonized zymosan, were incubated for 20 min at 37 "cwith M or 2 x M cysteine plus 1 0 - 4 ~HNE, added in succession. Cysteine strongly protected against the CL inhibition exerted by M HNE (Table 1). This protection, however, was never complete. InJEuence of Albumin. Previous studies done on the HNE-mediated effects on neutrophil functions such as migration or polarization were mostly performed in the presence of BSA.' The results obtained led some authors to suppose that the true active compound could be the adduct HNE-BSA. Another explanation might be, however, that BSA works by exhibiting the aldehyde to cells in the right geometry. CL experiments were generally done, for this reason, in the presence of 0.1 per cent BSA. To investigate the albumin role in our assays, some experiments were repeated by incubating the cells with 50 p1 of buffer (control) or 50 p1 of HNE M) before zymosan stimulation, the or BSA concentration was varied in the samples from 0 per cent to 2 per cent (w/v). We observed that CL production in both control and M HNE incubated cells was equally decreased by increasing BSA concentrations (Figure 6A), whereas M HNE showed a different behaviour (Figure 6B). Phagocytosis Assay
in order to understand the inhibitory effect exerted by high HNE concentrations on CL production, some experiments were carried out to evaluate the phagocytosis capability of the cells after expoor lo-' M HNE. sure to A strong reduction of phagocytosis caused by the aldehyde was observed (Figure 7); the result correlated well with the depression of CL previously observed.
150
C DI MAURO E T A L
A
0
10
20
time l m i n 1
-.E 5 0.5 1
0
-.-
20
10
time ( m i n )
Figure 1. Influence of HNE on CL production by unstimulated human PMN. Ten experiments in duplicate were performed by incubating cells with 50 g1 of different HNE concentrations or HBSS (control). Light emission was measured at 37 "C, every 60 s for 20 min. Background (-0.5 mV)was recorded using samples containing reagents alone, without cells. A typical experiment is shown. M HNE; lo-' M HNE; lo-* M HNE; + -A- Control; M HNE; --[I lo-' M HNE; -A10-9 M HNE.
DISCUSSION
-*
+
phi1 CL'* and to enhance CL production in stimulated PMN as shown by Van Epps and Garciai9, It is well known that HNE at micromolar concen- an increase in CL production by unstimulated and trations stimulates neutrophil migration. Since zymosan-stimulated cells after exposure to micromost of the chemotactic and chemokinetic agents, molar HNE concentrations was expected. In our especially peptides, are also able to induce neutro- experiments, however, the aldehyde did not signifi-
151
CHYDROXYNONENAL AND HUMAN NEUTROPHIL CHEMILUMINESCENCE
0
10
20
time ( m i n ) Figure 2. Influence of lo-' M M L P on PMN CL production. Ten cell preparations were tested by stimulating duplicate samples with 50 pl of MLP. The time-course of CL production in a single experiment is reported.
cantly increase the light emission. In consequence, it could be postulated that low HNE concentrations are involved in the induction of the inflammatory process through the stimulation of the migratory activity of PMN but not through the
enhancement of their oxidative metabolism. This effect is comparable to the observations made by Terui et a/.'' with Interleukin 1. This protein also was found to possess a chemotactic activity on PMN without inducing any enhancement of CL
'I -i
incubation time
. ... .. .
~
. .
.
.
. .. .
. .
4
.... .. ...... .* ... ....... . *. . .. .... . .... .. .. .. ..... ....
.. .
.' . . .
.. '. ..
.... . .... .,... ..... . .. . ...
.
. ..
.. ,. .. .' , . .
6
5
-log
,. ..
....
. . 7
8
9
HNE concentrations (MI
Figure 3. Influence of cell-HNE incubation time on CL production by zymosan-stimulated neutrophils. Samples prepared with eight different cell preparations were incubated for 0, 10,20 min with 50 pl of HBSS (control) or 50 pl of HNE (10-4-10-9 M) and then stimulated by opsonized zymosan. Results are referred to peak values S. E. as percentage versus control. Levels of significance were determined by Student's t-test.
152
C DI MAURO E T A L
A
0
10
20
time (min)
I
" 0
20
10 time
(min)
Figure 4. Time-course of CL production by HNE and zymosan stimulated PMN. CL was measured in eight different cell preparations which cells were incubated for 20 rnin at 37 "C in the absence (control) or in the presence of different HNE concentrations; then 50pl of opsonized zymosan were added. CL production was measured as mV every 60s for 20min. A M HNE; -0M HNE; -Arepresentative experiment is shown. -A- Control; M HNE; lo-' M HNE; -0- 10-8 M HNE; 10-9 M HNE.
+
+
production. On the other hand, it was confirmed by our experiments that fMLP was able to induce CL production in human PMN as well as to enhance the CL response by zymosan-stimulated PMN. Previous investigation^^*^^ indicated differences between HNE and fMLP stimulation on neutrophils. Our present results are consistent with the
+
supposition that these two classes of compounds act on PMN by triggering at least partly different mechanisms. High HNE concentrations inhibited neutrophil CL production and strongly reduced phagocytosis, these inhibitions not being related to a decrease in cell viability as indicated by the dye exclusion test.
153
4-HYDROXYNONENALAND HUMAN NEUTROPHIL CHEMILUMINESCENCE
0
lo
20
t ima
VOL.
8: 147-155 (1990)
Influence of 4-Hydroxynonenal on Chemiluminescence Production by Unstimulated and Opsonized ZymosanStimulated Human Neutrophils C. DI MAURO, G. CAVALLIT, M. C. AMPRIMOT, L. PARADISI, G. SCANOI, M. CURZIO AND M. U. DIANZANI Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy t Laboratorio di Chimica Clinica, Nuova Astanteria Martini, Piazza Donatori di Sangue, 10125 Torino, Italy
The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, Net-Leu-Phe ( N L P ) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between N L P and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different. KEY
wO~1)~--4-Hydroxynonenal; chemiluminescence; zymosan; neutrophils; respiratory burst.
INTRODUCTION 4-Hydroxy-2, 3-trans-nonenal (HNE) is one of the major products of peroxidative breakdown of arachidonic, y-linolenic agd linoleic acids.' It has been found to be formed during inflammatory and to possess a wide spectrum of biological effects on different cell types and rnolecules.'s4 In particular, in uitro studies showed that HNE in the micromolar range stimulates, in the presence of albumin, the morphological polarization and the oriented migration5 of rat polymorphonuclear neutrophils (PMN). In the case of human PMN it provokes a weak but significant stimulation of the random migration.6 In contrast, M HNE in uitro inhibits PMN-stimulated O i production,' neutrophil migration' and monocyte immune phagocytosis.* Considering the role
played by this aldehyde in phagocyte functions, the aim of the present study was to investigate its influence on the respiratory burst of unstimulated as well as of opsonized zymosan-stimulated human PMN, by means of the chemiluminescence technique in a luminol-amplified system. It is well known that when neutrophil plasma membranes interact with soluble or particulate stimuli, a complex series of events, resulting in a burst of oxidative metabolism associated with an increased glucose oxidation via the hexose monophosphate shunt takes place.' This process includes the generation of O;, other reactive oxygen radicals and H,O, which are produced by NADPH-oxidase and myeloperoxidase systems and that can be detected as light emission (chemiluminescence, CL). ' O - 1 1
Addressee for correspondence: Dr Clelia Di Mauro, Dipartirnento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Corso Raffaello 30, 10125 Torino, Italy. 0203 - 6484/90/030147-09%0.5.00 c1990 by John Wiley & Sons, Ltd.
148 MATERIALS AND METHODS Celt Preparation
Human polymorphonuclear leukocytes were harvested from heparinized peripheral blood of normal adult donors and separated by a modified Boyum's method.' Blood was mixed with 3 per cent dextran in 0-9 per cent NaCl solution. The red cells were allowed to settle for 60min at room temperature. The leukocyte-rich plasma obtained was removed and layered over a Lymphoprep gradient and centrifuged for 18 rnin at 800g to separate PMN. Residual red cells were removed by osmotic shock. PMN were washed twice in Hanks' balanced salt solution without phenol red (HBSS) and adjusted to 5 x lo6 ml-' in HBSS plus 0.4 per cent bovine serum albumin (BSA), whereas cells for phagocyte assays were diluted in buffer plus 0.2 per cent BSA. PMN were always more than 95 per cent of the leukocyte population. Cell viability was always over 94 per cent as judged by the Trypan blue exclusion test and it was not influenced by HNE exposure. In parallel malonaldehyde (MDA) levels were evaluated in blood samples, by the thiobarbituric acid test,I3 in order to investigate peroxidation reactions that might take place during the time of red-cell sedimentation. If so, lipid peroxidation end-products and in particular 4-hydroxynonenal, could influence the CL tests. Results obtained showed that a low level of MDA was produced. Values obtained were in accordance with the observations of Poli et aZ.14 who also demonstrated that 4-hydroxynonenal production was not detectable in red cells from normal subjects.
C DI MAURO E T A L
coupled to a computer (Olivetti M24) with a specific PHAGO-TEST program (LKB) for data integration. Results were represented on graphs with time units on abscissa and light intensity units (mV) on ordinate. The values in millivolts (mV) of peak height were evaluated and are reported in the results. The standard reaction mixture in the cuM Iuminol, vettes consisted of 600 p1 of 2 x 150 p1 of HBSS + 0-4 per cent BSA, 100 pI of cell suspension ( 5 x lo6 PMN ml-I). Cells were always preincubated for 5 min at 37 "C, then 50 pl of buffer (control) or HNE or Net-Leu-Phe (fMLP) were added and chemiluminescence was measured for 20 min. In other experiments cells were incubated for different times (0, 10, 20min) with the abovementioned substances and then they were stimulated by 50pl of serum opsonized zymosan; light emission was detected for 20 min. In addition, to evaluate the effects of SH-compounds in our experimental system, some assays were performed in the presence of cysteine and/or M HNE before zymosan stimulation. Other variations are indicated in the text and legends. Background emission (-0.5 mV) was recorded using samples containing reagents alone, without cells. The CL of resting PMN was obtained by measuring the light emitted by control cells without added stimuli. 4-Hydroxynonenal was a gift from Professor H. Esterbauer (University of Graz, Austria) and was prepared as previously described.s*l 6,1 Luminol and N L P were dissolved in dimethyl sulphoxide (DMSO) at a concentration of loe3 M. All compounds, before use, were diluted in HBSS to the desired concentration.
'
Zymosan Opsonization Zymosan opsonization was performed as previously described" by incubating at 37 "C, for 30 min, 80 mg of zymosan, 2 ml of human pooled AB serum and 2 mI of HBSS. The suspension was centrifuged at 800g for 10min. The pellet was washed twice with 0.9 per cent NaCl and resuspended with 4 ml of HBSS to a final concentration of 20 mg of zymosan m1-I.
Phagocytosis Assay
Samples containing 100 p1 of cell suspension were prewarmed at 37 "C for 5 min before adding 100 p1 of HNE (final concentration in the samples of or lo-' M) or 100p1 of buffer (control). Incubation with the aldehyde was carried out for 20 min, then 50 pl of opsonized zymosan (25,000 particles (mm3)- ') were pipetted into the samples. The reaction was allowed to proceed for 10min and then stopped by placing the tubes in ice. Slides Chemiluminescence Assay with cell suspensions were prepared and stained Measurements of chemiluminescence were per- with May Grunwald-Giemsa. Results were exformed with a luminometer (LKB Wallac 1251) pressed as percentage of phagocytosis (number of
CHYDROXYNONENAL AND HUMAN NEUTROPHIL CHEMILUMINESCENCE
phagocytosing cells (total number of cells)-' x loo). Chemicals
BSA, DMSO, fMLP, Luminol, Trypan blue and Zymosan were purchased from Sigma Chemical Co., St. Louis, MO, U.S.A. Dextran (p.m. 500 OOO) from Pharmacia, Uppsala, Sweden. Lymphoprep from Nyegard & Co. AS, Oslo, Norway. HBSS from Gibco Grand island, N.Y. Cysteine, malonaldehyde, thiobarbituric acid, were obtained from Merck Darmstadt, West Germany. RESULTS Chemiluminescence Assay Injuence of H N E and f M L P on C L Production by Unstimulated Neutrophils. HNE was tested in a wide range of doses (10-4-10-9 M); none of them was found able to enhance CL production by neutrophils (Figure lA, B). In some assays, CL measurements were prolonged up to 60 min to exclude the presence of a delayed peak, but in no case was an increase in CL detected. On the contrary, and lo-' M HNE decreased CL emission nearly to background values (Figure IA). In parallel, experiments were done by using, as a positive control, PMN in the presence of the soluble tripeptide fMLP at lo-' M. As shown in Figure 2 this peptide greatly increased PMN CL production, a peak of light emission being reached within 2 min. InJEuence of H N E and f M L P on C L Production by Opsonized Zymosan-Stimulated Neutrophils. The effect of HNE on zymosan-stimulated cells was evaluated by incubating samples at 37 "C for 0, 10, 20 min with 10-4-10-9 M HNE before adding zymosan (Figure 3). As it can be seen, and IO-' MHNE exerted a dose-related inhibition of CL production, maximal inhibition values being reached after 20min of incubation; lower HNE concentrations did not significantly modify detectable CL emission. in Figure 4A, B the time course of a typical experiment is shown. Zymosan stimulation of PMN resulted in a rapid increase in light emission reaching a peak within 10 min. HNE did not alter the time to reach the maximum. Cell stimulation with fMLP and zymosan added simultaneously, as
149
well as zymosan addition to cells previously incubated with the tripeptide for 20 min, resulted both in a shortening in the peak time and in an enhancement of CL produced with respect to cells stimulated by zymosan alone (Figure 5). Finally it should be noted that the extent of CL production varied greatly in both fh4LP or zymosan-treated PMN depending on the cell preparation tested. Injuence of Cysteine. The effect of thiols expected to act in uivo by detoxication of reactive aldehydes was also studied. The cells, before being stimulated by opsonized zymosan, were incubated for 20 min at 37 "cwith M or 2 x M cysteine plus 1 0 - 4 ~HNE, added in succession. Cysteine strongly protected against the CL inhibition exerted by M HNE (Table 1). This protection, however, was never complete. InJEuence of Albumin. Previous studies done on the HNE-mediated effects on neutrophil functions such as migration or polarization were mostly performed in the presence of BSA.' The results obtained led some authors to suppose that the true active compound could be the adduct HNE-BSA. Another explanation might be, however, that BSA works by exhibiting the aldehyde to cells in the right geometry. CL experiments were generally done, for this reason, in the presence of 0.1 per cent BSA. To investigate the albumin role in our assays, some experiments were repeated by incubating the cells with 50 p1 of buffer (control) or 50 p1 of HNE M) before zymosan stimulation, the or BSA concentration was varied in the samples from 0 per cent to 2 per cent (w/v). We observed that CL production in both control and M HNE incubated cells was equally decreased by increasing BSA concentrations (Figure 6A), whereas M HNE showed a different behaviour (Figure 6B). Phagocytosis Assay
in order to understand the inhibitory effect exerted by high HNE concentrations on CL production, some experiments were carried out to evaluate the phagocytosis capability of the cells after expoor lo-' M HNE. sure to A strong reduction of phagocytosis caused by the aldehyde was observed (Figure 7); the result correlated well with the depression of CL previously observed.
150
C DI MAURO E T A L
A
0
10
20
time l m i n 1
-.E 5 0.5 1
0
-.-
20
10
time ( m i n )
Figure 1. Influence of HNE on CL production by unstimulated human PMN. Ten experiments in duplicate were performed by incubating cells with 50 g1 of different HNE concentrations or HBSS (control). Light emission was measured at 37 "C, every 60 s for 20 min. Background (-0.5 mV)was recorded using samples containing reagents alone, without cells. A typical experiment is shown. M HNE; lo-' M HNE; lo-* M HNE; + -A- Control; M HNE; --[I lo-' M HNE; -A10-9 M HNE.
DISCUSSION
-*
+
phi1 CL'* and to enhance CL production in stimulated PMN as shown by Van Epps and Garciai9, It is well known that HNE at micromolar concen- an increase in CL production by unstimulated and trations stimulates neutrophil migration. Since zymosan-stimulated cells after exposure to micromost of the chemotactic and chemokinetic agents, molar HNE concentrations was expected. In our especially peptides, are also able to induce neutro- experiments, however, the aldehyde did not signifi-
151
CHYDROXYNONENAL AND HUMAN NEUTROPHIL CHEMILUMINESCENCE
0
10
20
time ( m i n ) Figure 2. Influence of lo-' M M L P on PMN CL production. Ten cell preparations were tested by stimulating duplicate samples with 50 pl of MLP. The time-course of CL production in a single experiment is reported.
cantly increase the light emission. In consequence, it could be postulated that low HNE concentrations are involved in the induction of the inflammatory process through the stimulation of the migratory activity of PMN but not through the
enhancement of their oxidative metabolism. This effect is comparable to the observations made by Terui et a/.'' with Interleukin 1. This protein also was found to possess a chemotactic activity on PMN without inducing any enhancement of CL
'I -i
incubation time
. ... .. .
~
. .
.
.
. .. .
. .
4
.... .. ...... .* ... ....... . *. . .. .... . .... .. .. .. ..... ....
.. .
.' . . .
.. '. ..
.... . .... .,... ..... . .. . ...
.
. ..
.. ,. .. .' , . .
6
5
-log
,. ..
....
. . 7
8
9
HNE concentrations (MI
Figure 3. Influence of cell-HNE incubation time on CL production by zymosan-stimulated neutrophils. Samples prepared with eight different cell preparations were incubated for 0, 10,20 min with 50 pl of HBSS (control) or 50 pl of HNE (10-4-10-9 M) and then stimulated by opsonized zymosan. Results are referred to peak values S. E. as percentage versus control. Levels of significance were determined by Student's t-test.
152
C DI MAURO E T A L
A
0
10
20
time (min)
I
" 0
20
10 time
(min)
Figure 4. Time-course of CL production by HNE and zymosan stimulated PMN. CL was measured in eight different cell preparations which cells were incubated for 20 rnin at 37 "C in the absence (control) or in the presence of different HNE concentrations; then 50pl of opsonized zymosan were added. CL production was measured as mV every 60s for 20min. A M HNE; -0M HNE; -Arepresentative experiment is shown. -A- Control; M HNE; lo-' M HNE; -0- 10-8 M HNE; 10-9 M HNE.
+
+
production. On the other hand, it was confirmed by our experiments that fMLP was able to induce CL production in human PMN as well as to enhance the CL response by zymosan-stimulated PMN. Previous investigation^^*^^ indicated differences between HNE and fMLP stimulation on neutrophils. Our present results are consistent with the
+
supposition that these two classes of compounds act on PMN by triggering at least partly different mechanisms. High HNE concentrations inhibited neutrophil CL production and strongly reduced phagocytosis, these inhibitions not being related to a decrease in cell viability as indicated by the dye exclusion test.
153
4-HYDROXYNONENALAND HUMAN NEUTROPHIL CHEMILUMINESCENCE
0
lo
20
t ima
