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Immunology |
Mestrado Acadêmico em Ciências Fisiológicas-CCS,4 Universidade Estadual do Ceará, Fortaleza-CE, Brazil
Depto de Fisiologia e Farmacologia, Faculdade de Medicina,5
Depto de Bioquímica e Biologia Molecular,6 Universidade Federal do Ceará, Fortaleza-CE, Brazil
Depto de Farmacologia,7 Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto-SP, Brazil
Instituto de Biomedicina de Valencia,8 C.S.I.C., Valencia, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNF by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.
macrophage activation, mast cells, neutrophil migration, porcine spermadhesin, PSP-I/PSP-II
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-6GalNAcß1-4GlcNAc-R motif [8], which block adhesive- and activation-related events mediated by CD22, further suggested a possible immunoregulatory activity for PSP-I/PSP-II. Recently, we reported that the spermadhesin PSP-I/PSP-II and its isolated subunits induced the recruitment of neutrophils into the peritoneal cavity of rats because of an indirect mechanism via the release of neutrophil chemotactic factors by spermadhesin-stimulated resident cells [9]. Thus, spermadhesin PSP-I/PSP-II might be regarded as an exogenous stimuli modulating the uterine immune activity to ensure reproductive success. Leukocyte activation by exogenous stimuli, or via cell-cell and cell-extracellular matrix interactions, generate cytokines, leukotriene B4, platelet-activating factor (PAF), C5a, and so on. Cytokines and leukotriene B4, mainly from a macrophage or mast cell origin, induce neutrophil migration both in vivo and in vitro and have been involved in aspects of the immune and inflammatory responses [10–12]. In the present study, we sought to investigate the participation of resident peritoneal cells (macrophages and mast cells) as the source of endogenous neutrophil chemotactic factors underlying the proinflammatory effect of PSP-I/PSP-II.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Wistar rats (weight, 150–250 g) were housed in a temperature-controlled room with free access to water and food. Investigations were conducted in accordance with current guiding principles for the care and use of research animals (NIH guidelines).
Isolation of Spermadhesin PSP-I/PSP-II
The PSP-I/PSP-II heterodimer complex was isolated by size-exclusion chromatography on Sephadex G-50 (Amersham Biosciences, Uppsala, Sweden) of the nonheparin-binding fraction from the seminal plasma of German Landrace boars as described previously [5]. The purity of the isolated protein was assessed by N-terminal sequence analysis (using an Applied Biosystems 473A instrument) and tryptic peptide mapping using an Applied Biosystems Voyager DE-Pro mass spectrometer (Langen, Germany) and sinapinic acid (saturated in 0.1% trifluoroacetic acid in 50% acetonitrile) as the matrix. Protein concentration was determined spectrophotometrically using its molar absorption coefficient (27 332 M-1 cm-1) [13].
Drugs and Reagents
Chemicals and biochemicals were of the highest purity-grade available and were purchased from the following companies: thioglycolate (Lab Difco Ltda, São Paulo, SP, Brazil), dexamethasone and indomethacin (Prodome Química e Farmacêutica, Campinas, SP, Brazil), and MK886 [L-663, 536 (3-[1-(4-clorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid) (Merck, Montreal, QC, Canada). Thioglycolate was solubilized in distilled water, dexamethasone in 0.1 M NaCl, indomethacin in Tris/HCl (pH 8.0), and MK886 in methyl-cellulose 0.1% aqueous solution. Antibodies against tumor necrosis factor (TNF) , interleukin (IL)-1ß, IL-4, and IL-10 were produced in sheep (National Institute of Biological Standard and Control, London, U.K.).
Isolation and Macrophage Monolayer Preparation
The method described by Cunha and Ferreira [14] was followed. Rats were injected i.p. with 10 ml of 3% thioglycolate. Four days later, the animals were injected i.p. with 10 ml of heparinized Roswell Park Memorial Institute (RPMI) 1640 medium. The peritoneal fluid was collected, and cells were washed twice with RPMI medium by centrifugation at 100 x g for 5 min. Pellets were resuspended in 1 ml of RPMI, and macrophages were counted. Macrophage monolayers were prepared by addition of 1 ml/well (106 macrophages) into 24-well culture plates. Macrophage adhesion was allowed to proceed for 24 h at 37°C in a 5% CO2 atmosphere. Thereafter, nonadhered macrophages were removed by three successive washes with RPMI. Plate-adhered macrophages were incubated with 1-ml solutions of PSP-I/PSP-II (1, 4, or 12 x 10-10 mol) for 15, 30, and 60 min at 37°C. The supernatants were discarded, and the macrophage monolayers were washed three times with RPMI. An additional 3-h incubation at 37°C with 1.5 ml of RPMI medium was performed for the release of chemotactic factors. Supernatants were collected, centrifuged at 100 x g for 5 min, and tested in the peritonitis model. Cell viability in every macrophage culture was analyzed by trypan blue staining.
Mast Cell Isolation
Adult rats were killed by cervical dislocation, and the peritoneal fluid was collected in aseptic conditions by flushing with 10 ml of RPMI medium (pH 7.2) containing 0.1% heparin. The peritoneal fluid was centrifuged at 146 x g for 7 min, and the pellet was resuspended in 1 ml of RPMI and then added into tubes containing 3 ml of 65% Percoll in Hanks solution supplemented with 5% bovine fetal albumin. After centrifugation at 400 x g for 20 min, the supernatants were discarded, and the pelleted mast cells were resuspended in 10 ml of RPMI and centrifuged at 146 x g for 7 min. This process was repeated three times, and the final pellet was then resuspended in 2 ml of RPMI for mast cell counting. Suspension containing 0.5 x 106 mast cells was stimulated with 12 x 10-10 mol of PSP-I/PSP-II for 1 h at 37°C and centrifuged at 146 x g for 7 min. The supernatants were removed, and 1.5 ml of fresh RPMI medium was added. After incubation for 1 h at 37°C, the supernatants were collected, filtered through Millipore SW INEX-13 no. 2 filters (Millipore, Sao Paulo-SP, Brazil), and used for incubation with macrophage monolayer cultures and for in vivo chemotactic activity test using the peritonitis model.
Dose-Response and Time-Course Neutrophil Chemotactic Activity of the Supernatant from the Macrophage Monolayer Stimulated by PSP-I/PSP-II and Assessed by the Peritonitis Model
A dose-response curve was constructed by incubating macrophages (cultured for 24 h) for 60 min in a 5% CO2 incubator at 37°C with 1 ml of RPMI per well containing 1, 4, or 12 x 10-10 mol of PSP-I/PSP-II. To the control group, RPMI was added in substitution of PSP-I/PSP-II. For the time-course curve, 1 ml of RPMI containing 12 x 10-10 mol of PSP-I/PSP-II was added to 24-h macrophage cultures and incubated for 15, 30, and 60 min at 37°C. Thereafter, the cells were washed three times with RPMI, and 1.5 ml of RPMI was added to each well, followed by incubation for 3 h in the same conditions. Supernatants were collected and injected i.p.. Evaluation of the neutrophil migration activity was done 4 h after injection using the peritonitis model. To this end, animals were killed and cells recovered by lavage of the peritoneal cavity with 10 ml of sterile saline containing 5 IU/ml of heparin. The fluid was collected for total and differential cell counts [9]. For total cell counting in a Neubauer chamber (Labor Optik, Shanghai, China), 20 µl of this fluid were diluted 1:20 (v/v) with Turk solution (Sigma, St. Louis, MO). For differential counting (neutrophils, eosinophils, and mononuclears), 30 µl were centrifuged at 400 x g for 10 min, applied to a glass slide, and stained with HEMA III. No discrimination between lymphocytes and monocytes was made. One-hundred cells were counted with an optical microscope using an immersion objective (100x). Results are expressed as the mean ± SEM of the number of cells per microliter of peritoneal wash of at least five different animals.
Effect of Administration of PSP-I/PSP-II and Supernatant of PSP-I/PSP-II-Stimulated Macrophage Monolayer in Animals Treated with Thioglycolate
To assess the effect of thioglycolate treatment on the neutrophil chemotactic activity induced by PSP-I/PSP-II and by the supernatant of PSP-I/PSP-II-stimulated macrophages, 1 ml of RPMI medium containing PSP-I/PSP-II (12 x 10-10 mol) and 1 ml of the supernatant obtained from 24-h cultured macrophages stimulated for 60 min with 12 x 10-10 mol of PSP-I/PSP-II were administered i.p. in normal rats and in rats that had been injected i.p. with 10 ml of 3% thioglycolate 4 days earlier to increase their peritoneal macrophage population [12]. Control animals were injected with 1 ml of RPMI medium (normal rats) or 1 ml of sterile saline (thioglycolate-treated rats). Neutrophil migration was evaluated in the peritonitis model, and the number of neutrophils in the peritoneal washes of control animals was subtracted from the number of neutrophils of rats injected with the spermadhesin or with the supernatant of PSP-I/PSP-II-stimulated macrophages.
Effect of Administration of Supernatant of Macrophages Stimulated with PSP-I/PSP-II in Animals Treated with Indomethacin, MK886, and Dexamethasone
Macrophages cultured for 24 h were stimulated for 60 min at 37°C with 12 x 10-10 mol of PSP-I/PSP-II, and 1 ml of the supernatants was injected into the peritoneal cavities of normal rats and of rats that had been treated 1 h earlier with MK886 (1 mg/kg; v.o.) and another group treated with dexamethasone (1 mg/kg s.c.) or 30 min earlier with indomethacin (5 mg/kg; s.c.). Control animals received only RPMI, and the onset of neutrophil migration was evaluated 4 h later.
Incubation of PSP-I/PSP-II-Stimulated Macrophages with Indomethacin, MK886, and Dexamethasone
Macrophages cultured for 24 h were incubated for 60 min at 37°C with indomethacin (10 µM), MK886 (1µM), dexamethasone (10 µg/ml), or RPMI, followed by incubation for 60 min with 12 x 10-10 mol of PSP-I/PSP-II. Cells were washed three times with 1 ml of RPMI and incubated with the drugs (without addition of spermadhesin) for 3 h at 37°C. Supernatants were collected and administered i.p. in rats. Neutrophil migration was determined and compared to that of animals that received the supernatant of nonstimulated macrophages.
Incubation of PSP-I/PSP-II-Stimulated Macrophage Cultures with the Supernatant of PSP-I/PSP-II-Stimulated Mast Cells
Isolated mast cells were stimulated for 60 min at 37°C with 12 x 10-10 mol of PSP-I/PSP-II, and the supernatant was added to 24-h cultured macrophages, followed by incubation for 60 min at 37°C. After this incubation period, 12 x 10-10 mol of PSP-I/PSP-II were added to each well and incubated for an additional 60 min. Cells were washed three times with 1 ml of RPMI. Then, 1.5 ml of RPMI was added, and cells were placed in a 5% CO2 incubator for 3 h at 37°C. One milliliter of supernatant was then injected i.p. into normal rats, and evaluation of neutrophil migration was assessed 4 h later. Groups of rats injected i.p. with either 1 ml of the supernatant of PSP-I/PSP-II-stimulated macrophages or 1 ml of RPMI were used as control.
Effect of Addition of Antibodies Anti-TNF and Anti-IL-1 on Neutrophil Migration-Inducing Activity of Supernatant of PSP-I/PSP-II-Stimulated Macrophage Monolayers
Monolayers of macrophages cultured for 24 h were stimulated for 60 min at 37°C with 12 x 10-10 mol of PSP-I/PSP-II. Supernatants (1 ml/well) were collected, and 50 µl of the anti-TNF or anti-IL-1 antisera were added 10 min before administration into the peritoneal cavity of normal rats. Evaluation of neutrophil migration was performed 4 h later and compared to the same activity of control groups injected with 1 ml of the supernatant of macrophages incubated with RPMI.
Incubation of PSP-I/PSP-II-Stimulated Macrophages with Supernatant of Mast Cells Stimulated with PSP-I/PSP-II Containing Antibodies Against IL-4 and IL-10
Mast cells were stimulated for 60 min at 37°C with 12 x 10-10 mol of PSP-I/PSP-II, and the supernatant was incubated for 10 min with solutions containing either anti-IL-4 (2.5 µg/well/1 ml) or anti-IL-10 (5 µg/well/1 ml) and then added to 24 h-cultured macrophage monolayers. After 1 h incubation at 37°C, 12 x 10-10 mol of PSP-I/PSP-II were added to each well and incubated for 60 min. After three washes with RPMI, 1.5 ml of RPMI was added and maintained in a 5% CO2 incubator for 3 h at 37°C. Supernatants were then collected and centrifuged for 7 min at 146 x g, and 1 ml of the final supernatants was injected i.p. into normal rats. Evaluation of neutrophil migration activity was done 4 h later. Control animals received either 1 ml of RPMI medium or the supernatant of PSP-I/PSP-II-stimulated macrophages incubated with preimmune serum.
Statistical Analysis
All results were expressed as the mean ± SEM for n = 5 experiments. Statistical evaluation was undertaken by ANOVA, followed by the Duncan test. A P value of less than 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Spermadhesin PSP-I/PSP-II stimulated the release by macrophage monolayers of a neutrophil migration-stimulating factor into the culture medium. This effect was time dependent, with maximal activity after 1-h incubation at 37°C. Figure 1 shows neutrophil chemotactic activity of supernatants of macrophage monolayers incubated for 60 min with increasing concentrations of PSP-I/PSP-II. We observed that 12 x 10-10 mol of PSP-I/PSP-II enhanced by 785% the migration of neutrophils to the peritoneal cavity of rats 4 h after i.p. injection compared with the activity of culture medium of nonstimulated macrophages (Fig. 1). The chemotactic factor was termed NCSMS (for neutrophil chemotactic substance derived from macrophages stimulated with spermadhesin PSP-I/PSP-II). When the clear supernatant of spermadhesin-stimulated macrophages was subjected to ultrafiltration using a 5-kDa cutoff membrane, the chemotactic activity was retained in the nonflow-through fraction, indicating that the molecular size of NCSMS was greater than 5 kDa.
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Thioglycolate Potentiates the Neutrophil Migration Induced by NSCMS
Intraperitoneal injection of 3% thioglycolate 96 h before the neutrophil migration assay increased by 106% the number of mononuclear cells in the peritoneal cavity (Fig. 2A). The neutrophil migration-inducing activity of both 12 x 10-10 mol of PSP-I/PSP-II and the supernatant of PSP-I/PSP-II-stimulated macrophages was potentiated by 176% and 149%, respectively, of the value obtained with control animals by previous treatment with thioglycolate (Fig. 2B).
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Dexamethasone, but not Indomethacin and MK886, Inhibited the Neutrophil Chemotactic Activity of NCSMS
Figure 3A shows the effect of the cyclooxygenase-inhibitor indomethacin, the leukotriene-inhibitor MK886, and the glucocorticoid dexamethasone injected 1 h before i.p. administration of the supernatant of PSP-I/PSP-II-stimulated macrophages on the neutrophil migration induced by NCSMS. Indomethacin and MK886 had no effect, whereas dexamethasone inhibited by 71% the number of neutrophils that migrated to the peritoneal cavity of animals. On the other hand, incubation of macrophage monolayers with indomethacin or MK886 before stimulation with PSP-I/PSP-II had no effect on the neutrophil migration-inducing effect of the supernatants, whereas this activity was reduced by 85% after pretreatment with dexamethasone (Fig. 3B).
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Effect of Supernatant of Mast Cells Incubated with PSP-I/PSP-II on Neutrophil Migration into the Peritoneal Cavity of Rats
The supernatant of mast cell cultures incubated with PSP-I/PSP-II did not induce neutrophil recruitment on injection into the peritoneal cavity of rats. In addition, i.p. administration of the supernatant of mast cell cultures, whether incubated or not incubated with PSP-I/PSP-II, 15 min before the i.p. injection of the supernatant of PSP-I/PSP-II-stimulated macrophages did not alter the neutrophil migration-inducing activity of NCSMS. However, preincubation for 60 min of macrophage monolayers with the supernatant of mast cell cultured in the presence of 20 x 10-10 mol of PSP-I/PSP-II before stimulation with the spermadhesin blocked by 74% the neutrophil migration-inducing effect of the supernatant of PSP-I/PSP-II-stimulated macrophages (Fig. 4).
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Antibody Anti-TNF Neutralized the Neutrophil Chemotactic Activity Induced by NCSMS
Addition of anti-TNF antiserum to the supernatant of PSP-I/PSP-II-stimulated macrophages before i.p. administration reduced by 68% the number of neutrophils in the peritoneal cavity of rats compared to control animals injected with preimmune, serum-treated NCSMS (Fig. 5A). On the other hand, an anti-IL-1ß antiserum had no effect on the neutrophil migration activity of the supernatant of PSP-I/PSP-II-stimulated macrophages.
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Antibody Anti-IL-4 Neutralized the NCSMS-Induced Neutrophil Migration-Inhibitory Effect of Mast Cell Supernatant Stimulated with PSP-I/PSP-II
The addition of an anti-IL-4 antibody to the supernatant of mast cells stimulated with PSP-I/PSP-II before the incubation with macrophage neutralized by approximately 70% the inhibitory activity of the supernatant of mast cells stimulated with PSP-I/PSP-II on the neutrophil migration-inducing activity of PSP-I/PSP-II-stimulated macrophages (compare Figs. 4 and 5B). This neutralization effect was not observed when the anti-IL-4 antiserum was replaced by anti-IL-10 antiserum or preimmune serum (Fig. 5B). Preimmune serum had no effect on the activity of the neutrophil chemotactic factor released by PSP-I/PSP-II-stimulated macrophages: i.p. injection of NCSMS (20 x 10-10 mol) previously incubated with preimmune serum induced recruitment of neutrophils into rat peritoneal cavity by 247% compared to the group injected with RPMI (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokines and leukotriene B4, mainly of macrophage or mast cell origin, induce neutrophil migration both in vivo and in vitro and have been involved in aspects of the immune and inflammatory responses [10–12]. Pharmacologic modulation experiments reported here (Fig. 3) show that dexamethasone, but not indomethacin or MK886, inhibited both the release of NCSMS into the supernatant of cultured macrophages and the neutrophil migration induced by NCSMS in rats that received an s.c. injection of 1 mg/kg of dexamethasone 1 h before i.p. administration of NCSMS. These features distinguish NCSMS from MNCF, a neutrophil chemotactic factor released by peritoneal macrophages in response to endotoxin [14]. The release of MNCF was inhibited by dexamethasone, but this glucocorticoid did not impair the activity of the chemotactic factor [15]. Our results, along with those of earlier reports concerning the inhibitory effect of dexamethasone on the release of cytokines by macrophages [15–18], pointed to the involvement of a cytokine as the NCSMS and strongly argued against the involvement of prostaglandins and leukotrienes. Furthermore, anti-TNF, but not anti-IL-1ß, neutralized the neutrophil chemotactic activity of the supernatant of PSP-I/PSP-II-stimulated macrophages (Fig. 5A), suggesting that TNF may be a relevant component of NCSMS. Disclosing the mechanism used by this cytokine needs further investigation.
We also investigated the possible involvement of mast cells in the release of the chemotactic factor induced by PSP-I/PSP-II. The supernatant of mast cells cultured in the presence of spermadhesin PSP-I/PSP-II was devoid of neutrophil chemotactic activity to the peritoneal cavity of rats, further indicating that NCSMS had a macrophage-specific origin. Moreover, i.p. injection of PSP-I/PSP-II-stimulated mast cell culture before administration of the supernatant of PSP-I/PSP-II-stimulated macrophages did not alter NCSMS activity. However, incubation of cultured macrophages with the supernatant of spermadhesin-stimulated mast cells before stimulation with PSP-I/PSP-II inhibited the chemotactic activity of the macrophage cell culture medium (Fig. 4). Our interpretation is that spermadhesin-stimulated mast cells released a negative modulator of the secretion of the chemotactic factor by macrophages. This conclusion is in line with the observation that depletion of peritoneal mast cells by chronic treatment with compound 48/80 or by i.p. administration of water did not alter the onset of neutrophil migration induced by fMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) or PSP-I, which act as direct inducers of neutrophils [9]. In addition, animals injected with the supernatant of spermadhesin-stimulated mast cells before the administration of fMLP did not impair the neutrophil chemotaxic activity of this tripeptide (data not shown). Anti-IL-4, but not anti-IL-10, blocked the neutrophil migration-inhibitory activity secreted by PSP-I/PSP-II-stimulated mast cells, pointing toward IL-4 as a major player in the mast cell NCSMS-modulation activity.
In conclusion, our data support the role of spermadhesin PSP-I/PSP-II as a modulator of the uterine immune activity, which may contribute to reproductive success in the pig. In a more general sense, the present study shows the importance of mast cells in the modulation of the neutrophil-migration response triggered by activated macrophages. The emerging view is that the balance between cytokines released by macrophages (TNF) and mast cells (IL-4) appear to control the onset of neutrophil migration in response to inflammatory stimuli, thereby avoiding excessive neutrophil recruitment. This regulation seems to be crucial during situations in which the excessive accumulation of neutrophils would lead to tissue damage (i.e., in postmating endometritis, the pathogenesis of human rheumatoid arthritis, and so on). Thus, in addition to its physiological role, spermadhesin PSP-I/PSP-II might represent a useful tool for dissecting the biochemical details of macrophage-induced inflammation and its regulation by mast cells.
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FOOTNOTES |
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2 Correspondence (Spain): Juan J. Calvete, Instituto de Biomedicina, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. FAX: 34 96 3690800; jcalvete{at}ibv.csic.es
3 Correspondence (Brazil): Ronaldo A. Ribeiro, Depto de Fisiologia e Farmacologia, Universidade Federal do Ceará, R. Cel. Nunes de Melo, 1127-Rodolfo Teófilo, 60 430.270 Fortaleza, CE, Brazil. FAX: 55 85 2888333; ribeiror{at}ufc.br
Received: 13 November 2002.
First decision: 28 November 2002.
Accepted: 9 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and leukotrienes. Infect Immun 1997 65:2740-2746[Abstract]This article has been cited by other articles:
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