Production of nitric oxide (NO) in human hydatid,3sis., " Relationship between nitrite production and interferon-y evels. Chafia Touil-Boukoffa "~, Brigitte Bauvois ...
Biochimie (1998) 80, 739-744 O Soci6t6 franqaise de biochimie et biologic mol6culairc / Elsevier, Paris
Production of nitric oxide (NO) in human hydatid,3sis.," Relationship between nitrite production and interferon-y evels Chafia Touil-Boukoffa "~, Brigitte Bauvois b, Josiane Sanc6au b, Boussad HamriouP, Juana W:~etzerbin b* "Laboratoire de Biochimie, ISN-USTHB, Universit~~Bab-Ezzouar Algiers. Algeria hUnitd 365 INSERM, Institut Curie, Pavilion Pasteur, 26, rue d'Uhn,75231 PalTs cedex 05, France
(Received 20 May 1998; accepted 9 July 1998) Abstract -- Human hydatidosis is characterized by a prolonged coexistence of parasite (Echinococcus gramdosus) and host without
effective rejection. The basis of the immune response of the patient is poorly understood. Previously, we reported the presence of IFN, TNF-a and IL-6 activities in the serum of patients with liver and lung hydatidosis. In the present work, we have investigated the production of nitrite (NO2-) in the serum of hydatidic patients carrying hepatic and pulmonary cysts (range 36-300 [.tM). Our present data show a correlation between the production of nitrite + nitrate (NO2 + N O 3 ) and that of circulating cytokines IFN and IL-6. In relapsing patients who did not produce IFN and IL-6, the observed serum NO z- concentrations were low (range 10-37.2 [aM), as compared to those detected in patients before surgery. Induction of NO ,,ynthase in,'.eukocytes from hydatidic patiems was induced by stimulating these cells with a specific parasitic antigen, Antigen-5, as assessed by the increased levels of N O 3 + N O 2 in the range of 60-85 ~tM for patients with liver hydatidosis, as compared to the 20-25 ~tM detected in healthy controls. Collectively, our data indicate that NO.-, + NO~ levels correlate with IFN levels and immunoreactivity, and overall suggest that IFN-y and nitric oxide productior~ together play a role in the host defense mechanisms in human hydatidosis. © Socirt6 franqaise de biochimie et biologic molfculaire / Elsevier, Paris nitric oxide I hydatidosis I interferon gamma
I. Introduction
Nitric oxide (NO) from activated murine macrophages is an important cytostatic and cytotoxic rnediator for tumor cells and Ibr a variety of pathogens including fungal, helminth, bacterial and protozoan agents !1, 21. In murine macrophages, NO synthase activity is inducible by LPS and some cytokines like IFN-y and TNF-ct [31. NO pathways have been characterized in human eosinophils [41, human monocytes [5, 61, B lineage cells !171and acute myelogenous leukemia ceils [8j. Several recent studies attribute an important role to reactive nitrogen intermediates in antimicrobial activities of macrophages [3, 91. Human NO synthase expression during parasitic elimination has been reported by several authors. The role of NO in the killing of parasites by infected human macrophages was demonstrated in the case of Leishmania 12] and Tpw.panosoma cruzi [ lOl. Patients with Plasmodium falciparum or Plasmodium vivax malaria also * Correspondence and reprints Abbreviations: IFN-y, interferon gamma; IL-, interleukin; NO, nitric oxide; NOz + NO3, nitrite + nitrate; I NF-~t, tumor necrosis factor alpha.
show increased nitrite and nitrate plasma levels [lll. However, additional in vivo investigations in humans are required to delineate more precisely the role of NOmediated effects in immune responses during parasitic infections. Hydatidosis constitutes a major health problem in North Africa. It is a form of parasitosis vectored in man by a larva stage cestode Echinococcus granulosus. This parasitosis is characterized by a prolonged coexistence of parasite and host without effective rejection !121. However, the basis of the cellular immune response of the patients is poorly understood. We have previously shown the presence of TNF-ct, IL-6 and IFN activities (mixture of IFN-c~, -~ and -y, range 32-500 U/mL) in sera of hydatidic patients exhibiting hepatic and pulmonary cystic localization, and this production correlated with immunoreactivity versus parasitic antigen (Antigen 5)I131. In the present work, we have investigated the production of NO in hydatidic patients by measuring the concentration of NO 2- + NO 3- in sera. We have also measured the levels of NO 2- + NO3- and IFN-y released by leukocytes stimulated in vitro with the major parasitic antigen (Antigen-5) in relation to the clinical stage of the patients.
740 2. Materia|s and methods
2.1. Patients All patients were admitted to the Mustapha Bacha Hospital (Department of Surgery, Algiers, Algeria). The mean age of the patients was 32 + 10 years, 2/3 were male and they did not present other parasitic or bacterial infections. None of the subjects had ever been previously transfused and r~o pharmacological treatment was given before bleeding manifestations occurred. The patients did not show urinary dysfunction and they had low exogenous dietary nitrate ingestion. Serological reaction against parasitic antigen (Antigen-5) was tested in each case by immunoelectrophoresis. The diagnosis was surgically confirmed in all cases. Healthy controls (n = 17, from the same Algerian region) were adult volunteer blood donors (4(~ ~ .~0 years) none of whom showed positive immunoreactivity by immunoelectrophoresis using Antigen 5. Two groups of Algerian patients were tested: the first group was composed of 92 hydatidic patients with different cystic localizations and in the presurgical stage. The patients were tested for circulating nitrate/nitrite levels and for their immunoreactivity against Antigen-5. Eighteen of these patients with liver hydatidosis were also tested before surgery (1 week before surgical cyst removal) for circulating nitrite, IFN, TNF-ct and IL-6, for CD8/CD4 T cell ratios and for in vitro induction of IFN-y by Antigen-5: two of them relapsed 18 months after surgery. The second group was composed of eight individuals and the production of IFN-y and NO secretion in supernatants of leukocyte cultures stimulated by Antigen 5 was analyzed. 2.2. Antigen-5 preparation Antigen-5 was prepared as reported previously by chromatography on Sephadex G-200 1131. Eluted peaks were detected by spectroscopy recording at 280 nm and were analyzed by immunoelectrophoresis using a specific rabbit hyperimmune serum against anti-Antigen-5 for the detection. Antigen-5 was localized in the second eluted peak corresponding to a molecular mass of about 65 kDa [121, The eluted fractions were pooled, lyophilized and used for serological tests and for the induction of IFN and NO production. 2.3. Leukocyte preparation and cell cultures Leukocytes were prepared from peripheral blood of patients and healthy donors. Fifty mL of peripheral blood were collected in preheparinized syringes. Leukocytes were collected after sedimentation in Dextran 500 in 0.9% (w/v) NaCi. Lymphocytes were separated by FicollHypaque density (1.077 mg/mL) gradient centrifugation (Sigma, Saint Louis, USA). The mononuclear cell fraction
TouiI-Boukoffa et al. at the interface was collected and resuspended in serum free-medium. Cell viability was assessed by trypan blue dye exclusion (viability = 98%). Cells were washed twice with PBS and adjusted to 5 106 cells/mL in RPMI 1640 supplemented with 15 mM "Iris, pH 7.5, 2 mM glutamine, 5% fetal calf serum, 100 IU/mL penicillin, 100 lag/mL streptomycin and 10 lag/mL gentamicin. Cells were then cultured in the absence or presence of increasing concentrations of Antigen-5 (from 0.2 to 25 lag/mL) and incubated in Belco spinners flasks at 37 °C for 18-20 h. Cells were then centrifuged at 10 000 g for 15 min and supernatants were collected and stored frozen a t - 7 0 °C until use or processed immediately. 2.4. Nitric o',ide assay NO production in serum of patients or in supernatants of stimulated leukocytes was assessed by determination of the end products of NO oxidation. Total NO2- + NO3-concentration was quantified by the Griess reaction [14]. NO 2- was assayed after reduction of NO 3- by nitrate reductase-containing Pseudomonas oleovorans bacteria (ATCC 8062). Briefly, I00 laL of each serum or supernatant to be tested was mixed with 50 laL of a 1/10 dilution of pelleted bacterial cells. In parallel, NO2- standards for calibration curves were treated similarly as the samples. More than 90% of NO 3- was converted to NO_,- under these ~,onditions. After incubation at 37 °C for 90 min, the samples were centrifuged and mixed with 1001aL of Griess reagent (0.5% N-I-naphthyi ethylene diamine in 20% HCI, 5% sulfanilamide in 20% HCi (! v/v). After 20 rain incubation at room temperature, the formation of the chromogenic derivative was detected by spectrophotometry at 540 nm. Sodium NO~ standards were run along with the experimental samples for the calibration of the test. 2.5. IFN biological assays IFN activity in the serum of the patients was assayed by a cytopathogenic inhibition test, using Wish cells infected with vesicular stomatis virus (VSV) as indicator system. Briefly, Wish cells (16 x 10 3 per well) were seeded in 96-well microplates (Nunclon) in 100~tL of Eagle's minimal essential medium supplemented with 10% fetal calf serum. After 24 h, the medium was replaced by 100 laL of various dilutions of the samples to be tested or with various IFN dilutions for the calibration curve. After another 24 h, plates were drained and 100 pfu of VSV were added in 100 ~tL medium containing 5% fetal calf serum. A sample of IFN international NIH references was incubated in each assay in parallel. Titers were expressed in international reference units as the reciprocal of dilution giving 50% of the cytopathogenic effect I151. In order to identify the type of IFN detected, neutralization assays were carried out by incubating the samples
NO production in human hydatidosis for 2 h at 37 °C with an excess of neutralizing anti-lFN antibodies and then assayed for residual IFN activity as described above. Rabbit polyclonal antibodies against human recombinant IFN-a and human recombinant IFN-y prepared in INSERM Unit 365 (Paris) by injecting recombinant IFN-~ or IFN-y, exhibited neutralization titers of 40 000 against 10 units of the corresponding IFN. Antihuman IFN-['5 was kindly provided by Dr. P. Pitha-Rowe (Johns Hopkins University, Baltimore, USA) and had a neutralization titer of 100 000 against 10 units of IFN-~.
2.6. TNF-tt biological assay TNF-ct activity was tested by measuring its cytotoxic activity on L929 cells in the presence of 1 ~tg/mL of actinomycin D (Sigma Saint-Louis, USA), as previously reported i l 6]. A dilution of a recombinant TNF-~ sample (2000 U/mL, Genentech, provided by BoehringerIngelheim, Vienna, Austria) was used as reference. A rabbit antiserum against recombinant TNF-ct (neutralization titer 30 000 against 10 U/mL of TNF-a), prepared in INSERM Unit 365 (Paris), was used to assess the specificity of the detection.
741 Table I. Sera NO 2 + NO~ ~evels in patients w'ith hydatidosis.
Sera n = 73
Cystic localization Liver
Clinical state None*
n=5
Liver
n n n n
8 3 1 1
Liver Lung Brain Brain
n= !
Spleen
Relapsing patients ~ None* None* None* None* (with increased eosinophils) None* (with calcified cyst)
= = = =
Immum~- NO 2 +NO reactivity (/~M+__SD) 105 +_7.1 17+_2.5 +
7.1 57.8 + 6.6 24
-
i 23.6
-
Healthy controls n = 17
59.1 +
26.2
37.2 +_ 1.5
*Not surgically treated; "Patients who relapsed after 1 year to 18 months of radical surgery: +, positive serologic reaction.
2.7. IL-6 biological assay IL-6 activity was quantified on 7TDI hybridoma cells as a function of its growth factor activity, as described by Van Snick et al. [I 7]. A sample of recombinant IL-6 (2000 U/mL) was used as ~eference in each assay. A monocionai antibody against recombinant IL-6 (neutralization titer I0 000 against IL-6 (gift from Dr. J Wijdenes, Besanqon. France) was used for detection.
2.8. Flow cytometo' analysis All staining steps were performed in PBS supplemented with !% BSA aod 0.1% sodium azide and were carried out in 96-microwell plates. Cells to be stained (2 × IOs) were pelleted by ccntrifugation, suspended in the appropriate concentration of antibody (CD4 mAb vn'c or CD8 mAb vn'c from Immunotech) and incubated on ice for 30 rain. After incubation, cells were washed twice and fixed with 1% paraformaldehyde in PBS. Detection was performed on a FACS analyzer (Becton-Dickinson, Mountain View, CA, USA); 10 000 events were recorded and analyzed using Lysys software (Becton Dickinson). Fluorescence data were expressed in relative fluorescence intensity (%) and antigen relative density per cell was obtained by subtracting the peak channel number of the negative control from the peak channel number of the corresponding experimental sample. 3. Results
and
discussion
3. I. Detection of N O r in sera of hydatidic patients with d~fferent ~Lvstic localizations As shown in figure !, sera of presurgicai hydatidic patients were found to contain significantly higher NO~ +
NO 3 levels (96 + 60, n = 88) than sera from healthy controls (37 + 6, n = 17) and from relapsing patients ( 17 _+5.5, n = 5). In all cases, the major oxidation product of NO was found as NO,-. When analyzed more closely. patients with liver hydatidosis and immune reactivity towards Antigen-5 had the highest serum NO 2- levels. Indeed, patients with liver hydatidosis displayed higher serum levels of NO3-/NO 2 when their immune reactivity toward Antigen-5 was detectable (median 75) than patients not displaying such immunoreactivity (median 42.4) (table !). One patient who developed in parallel brain hydatidosis and had increased circulating eosinophii levels, exhibited a very high concentration of serum NO, ( 123 ~tM; table 1). In contrast, patients who relapsed after ! year and patients with calcified splenic cysts showed low NO,-levels (26.2 l.tM; table I). These results suggest that, in addition to the clinical stage, cystic localizations and associated disorders may contribute to the observed NO synthase induction. A similar observation was previously reported for Tanzanian children with malaria [18] where an inverse relationship between malaria severity and NO synthase 2 expression was suggested.
3.2. Antigen-5 mediated production of IFN-y We have studied the in vitro production of IFN-y in 16 patients from the above group, two of them relapsing 18 months alter surgical cyst removal. IFN-y synthesis was previously found to be related to the clinical stage of this disease [13]. In preliminary experiments, we found a maximal induction of IFN-~, for leukocyte stimulation by Antigen-5 concentrations from 2 to 20 ~tg/mL, both tbr
742
Touil-Boukoffa et al.
300
250 0 0
~
2o0 0
000
-' Z +
150
•-~ omt Z
iO0
o~
o
50 ooo 0
Controls n= 17
Patients n=88
leukocytes collected from patients betbre or after surgery (data not shown). Therefore, the dose of 10 ~tg/mL for Antigen-5 was chosen in following experiments. Analysis of IFN production in patients revealed a marked difference from that of healthy controls (table il). IFN titers were in the range of 32-100 U/mL (median --- 64) before surgery and 8-32 U/mL (median 32) after surgical cyst removal (table !i). Following surgery, IFN-',/leukocyte production was low and in some cases barely significant (table II) suggesting possible differences of degree of sensitization of T cells to Antigen-5. T cells of the 16 patients displaying immunoreactivity versus antigen, particularly before cyst eradication, still responded to the antigenic restimulation after undergoing surgical cyst removal (table I!), Interestingly, the two relapsing patients did not respond to antigen stimulation and their leukocytes did not secrete significant amounts of IFN-7 when stimulated with Antigen-5, whereas a marked increase in the CD8/CD4 T cell ratios was observed (table IIL Altogether, these preliminary observations suggest a CD4 + dependent response in these patients. Moreover, our results indicate that the reduction in IFN-'f titer correlates with the lack of immunoreactivity following surgery. This may be explained by the fact that surgical cyst removal reduces the availability of parasitic soluble antigen. In contrast, the lack of IFN-'~ induction in relapsing patients may be associated with the significant decrease in CD4 ÷ cells.
Relapse 0-5
Figure 1. N O 2- + N O 3" levels in sera of hydatidic patients. NO2 + NO3 levels in sera of healthy controls, hydatidic patients before surgery and relapsing patients were measured as described in Materials and methods. Statistical analysis of the results (mean + SD): controls, 37.18 + 1.51; patients, 96.02 + 6.41; relapse, 16.98 + 2.48. With regard to the P test studient, P = 0.0001 for controls versus patients, P = 0.024 for controls versus relapse and P = 0.0037 for patients versus relapse.
3.3. NO 2- and cytokine levels in sera o.f patients with liver hydatidosis Circulating serum levels of cytokines (IL-6 and TNF-u) and NO2 were also measured at the same period in the patients tested for in vitro production of IFN-~,. Analysis of circulating cytokine levels and serum NO2levels revealed distinct patterns in these patients as compared to normal donors (table II). The clinical stage appeared to influence both IFN, IL-6 and NO production and serological reaction against Antigen-5 (table IlL As reported above, NO 2- levels were high in presurgical patients, and very low in relapsing patients not reactive for Antigen-5 (table 11). The correlation observed between IFN, IL-6 and NO2-levels and serological reaction against the major parasitic antigen indicates that all these parameters are related and involved in the immune response in liver hydatidosis. All patients tested exhibited increased ratios of CD8/CD4 T cells (table H) thus suggesting that in this disease, as in many other parasitoses, CD8 cells represent the major T cell population. Interestingly, we observed a significant increase in CD8/CD4 accompanied by a marked drop in NO 2- levels in serum of relapsing individuals who did not display immunoreactivity versus parasitic antigen (table I). In this group of relapsing patients, it may be concluded that the lack of IFN (IFN-~,) is associated with a reduction in NO2-prodt, ction and with
NO production in human hydatidosis
743
Table 11. CD8/CD4 T cell ratios, cytokine production and sera NO2- levels in patients with liver hydatidosis. Patients
Clinical stage
CDS/CD4 + SD
IFN U/mL + SD
7NF-ct U/mL ± SD
IL-6 U/mL ± SD
NO, +NO~ uM ± SD
n = 16
b
2.6 +_0.03
60.8 _+ 10.632-100"
50.3 + 5.2
97.3 + 29.6
152.8+ 12.2
+
n = 16
a
3.1 ± 0.05
~ -, 2.48-32*
12.5 + 1.0
5g.3 ~ ~
N.D.
+
n -- 2
R
4.4-8.5
0-40*
64
0
152.8 + 12.2
-
0.46 + 0.02
7.5 + 4.4
5.6 + 1.4
3.3 +_2.5
37.7 + 6.9
-
Healthy controls n =3
lmmunoreactivity
b, Before surgical removal (1 week); a, after surgical removal 172 h); R, patients who relapsed after 18 mcnths of surgery; *IFN produced in vitro by lymphocyte T cultures prepared from patients and stimulated with 10 ~,tgof Antigen-5; N.D., not determined; +, positive serological reaction against Antigen-5.
patients prescn'ing both liver hydatidosis and asthma before (283-315 jaM) and after (261-271.5 gM) leukocyte stimulation with Antigen-5, Banes previously reported that levels of exhaled NO were markedly elevated in patients with asthma without glucocorticoid treatment [19]. This latter observation suggests that in addition to the stimulatory action of parasitic antigen, additional mechanisms are probably implicated in triggering NO synthase induction.
a decreased number of CD4 + cells. Whether IFN-y is directly involved in CD4 + T cell depletion remains to be demonstrated, 3.4. NO 2- and IFN ~: production by ieukocytes o f hydatidic patients
NO2 + NO 3- and IFN-y productions were measured in leukocyte culture supernatants from hydatidic patients (table IH). Again, the major oxidation product of NO was found as NO2- (table I!I). When 14 patients were coanalyzed for NO,- and IFN-y, a significant correlation was found between NO:,- levels and production of IFN-y (ligure 2). In parallel, non-significative levels of IFN (0-4 U/mL) and NO2 + N O 3 (22-29 taM) were detected in the supernatants of healthy doi~ors. It was found that leukocytes of patients with liver hydatidosis, in addition to the production ot" IFN, responded actively to the antigen stimulation by an elevated release of N O , + N O 3 (82-116 gM) as compared to healthy controls 1,22-29 taM) and to patients with lung hydatidosis (31-44 taM) (table lll).lnterestingIy, very high and similar levels of NO.,+ NO a- were detected in leukocyte supematants of two
4. Concludh~g remarks Collectively, the present study is the tirst to provide evidence that NO is actively produced during the course of Echim~coccus gramdosus human infection and that its secretion correlates with !FN-(production and varies according to cystic localization (in the lung or in the liver), clinical state and imrnunoreactivity against parasitic antigen. Whether NO synthase triggering in leukocytes of hydatidic patients plays a role in the clinical course of this disease remains hov.ever to be demonstrated.
Table III. NO2 and IFN-y in supernatants of leukocyte cultures in hydatidic patients. NO, (~lM) Samples
Cystic localization
NO_,+NO.¢ iBM)
.
.
.
.
.
.
.
.
.
.
.
.
.
+ .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+
IFN-}," (U/mL)
.
n=2
Lung
!1-12
25-35
14-15
31-44
8
n=4
Liver
4!-55
61-85
70-73.5
82-116
16-32
n=2
Liver*
255-258.5
298.8
261-271.5
283-315
32-64
9-14
20.5-25
11- i 8
22-29
0-4
Healthy controls (n = 3)
Non-treated (-) or treated (+) patients with Antigen-5; *patients with liver hydatidosis and asthma; 'qFN-y produced in vitro in leucocyte cultures stimulated with Antigen-5.
744
TouiI-Boukoffa et al. 80 o
60
E 40 QQ
"
20
0
1)
100
200
300
4011
Nitrite + Nitrate (HM) Figure 2. Correlation between NO_, + NOs- levels and IFN-y in supernatants of leukocyte cultures from hydatidic patients, Supematants of patient leukocytes (n = 14) stimulated in vitro with Antigen-5 were assessed for NO 2- + NO.a levels and IFN-y activity, as described in Materials and methods, R 2 = 0.961. Non-significant levels of IFN (0-4 U/ml) and NO2 + NO 3 (22-29 ~tM) were detected in the supernatants of healthy donors.
Acknowledgments The authors thank the clinicians of the Service de Parasitologie, lnstitut Pasteur, Alger and the clinicians of the Service de Chirurgie (CHU, Alger) for providing serum samples. We are grateful to Dr. Jean-Pierre Kolb for critical reading of the manuscript and helpful discussions, Catherine Silvestri for technical assistance and lsabelle Ricard for secretariat assistance. This work was supported by the Frencll Ministry Ibr Foreign Affairs (93 Mars 28 program) and the INSERM.
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