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-primed-macrophages
* Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil;
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Japan;
Departamento de Imunologia, Universidade de São Paulo, Ribeirão Preto, Brazil; and
Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brazil
Correspondence: Dr. Mauro M. Teixeira, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627–Pampulha, 31270-901 Belo Horizonte, MG, Brazil. E-mail: mmtex{at}icb.ufmg.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inducing agents but not in naïve mice. Maximal recruitment was observed at a dose between 250 and 1250 ng tGPI-mucins. There was a significant elevation in the levels of MCP-1 in the pleural cavity of primed animals injected with tGPI-mucins, and in vivo neutralization of MCP-1 abolished leukocyte recruitment. Pretreatment with anti-MIP-1
or anti-RANTES had no effect on the recruitment induced by tGPI-mucins. MCP-1 immunoreactivity was detected in pleural macrophages, and macrophages produced MCP-1 in vitro, especially after priming with IFN-. Finally, tGPI-mucins induced significant leukocyte recruitment in primed C3H/HeJ but not in TLR2-deficient mice. Together, our results suggest that T. cruzi-derived GPI-mucins in conjunction with IFN- may drive tissue chemokine production and inflammation and bear a significant role in the pathogenesis of Chagas disease.
Key Words: cell trafficking • inflammation • inflammatory mediators • chemokines • protozoan parasites
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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More recently, we demonstrated that glycosylphosphatidylinositol-anchored mucins-like glycoproteins from Trypanosoma cruzi trypomastigotes (tGPI-mucins) or highly purified glycosylphosphatidylinositol from tGPI-mucins (tGPI) activate macrophages in vitro to produce the proinflammatory cytokines tumor necrosis factor (TNF-) and interleukin (IL)-12 as well as nitric oxide (NO) [6
7
8
9
]. Of interest, these two cytokines and NO are of crucial importance for the control of T. cruzi replication in vivo [5
, 10
, 11
]. In addition, we have shown that in vitro exposure of macrophages to live trypomastigotes or tGPI-mucins leads to expression of different chemokine mRNAs as well as proteins [12
13
14
]. The ability of tGPI-mucins to promote leukocyte recruitment in vivo has not been shown.
The paradigm for the recruitment of leukocytes into tissues predicts an essential role for the activation of G-protein-coupled serpentine receptors on the leukocyte surface. Chemokines can trigger such receptors and have been shown to mediate the recruitment of a range of leukocyte subsets in vivo [15
]. Recently, we have shown that chemokines are expressed during the course of the experimental T. cruzi infection and play a role in activating interferon- (IFN-)-primed macrophages to kill T. cruzi trypomastigotes in a NO-dependent manner [13
, 15
]. Of note, the chemokines macrophage-inflammatory protein-1 (MIP-1), regulated on activation, normal T expressed and secreted (RANTES), and especially monocyte chemoattractant protein-1 (MCP-1) were shown to be the most active in inducing parasite killing [12
].
Here, using the pleural cavity of mice as a reproducible model for the evaluation of leukocyte recruitment in vivo, we demonstrate that tGPI-mucins are highly effective in promoting cell recruitment and inflammation by activating Toll-like receptor (TLR)2 in animals primed with IFN--inducing agents. Further, the levels of MCP-1 in the pleural cavity were elevated, and MCP-1 immunoreactivity was detected in macrophages of primed animals injected with tGPI-mucins. Finally, the tGPI-mucins-induced, in vivo leukocyte migration was abolished by in vivo neutralization of MCP-1. Together, our results are consistent with the hypothesis that tGPI-mucins and endogenous IFN- may act together to promote inflammation during infection with T. cruzi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/- (C57BL/6), CXCR2-/- (BALB/c), and MIP-1-/- (C57BL/6) mice and their respective wild-type controls were purchased from The Jackson Laboratory (Bar Harbor, ME). TLR2-/- mice (129-C57BL/6) were produced as described previously [16
]. Animals were bred and maintained in our Bioscience Unit.
Reagents and antibodies
Methylated bovine serum albumin (MBSA), concavalin A (Con A), lipopolysaccharide (LPS; 011:B11), o-phenylenediamine (OPD), and Freund’s complete adjuvant (FCA) were purchased from Sigma Chemical Co. (Poole, UK); Dulbecco’s phosphate-buffered saline (PBS; calcium- and magnesium-free, pH 7.4) came from Life Technologies (Paisley, UK). IL-12 was a gift from Genetics Institute (Boston, MA). Specific rabbit polyclonal antibodies, anti-MIP-1, anti-RANTES, and anti-MCP-1 have been tested previously in mice [17
, 18
] and were a kind gift of Dr. N. W. Lukacs (University of Michigan, Ann Arbor).
Purification of tGPI-mucins
tGPI-mucins were isolated from T. cruzi trypomastigotes as described previously [6
7
8
]. The nonglycosylated lipids were removed by three consecutive extractions with chloroform and methanol. The tGPI-mucins were then extracted by chloroform, methanol, and water followed by sequential butanol:water partition and purification by hydrophobic-interaction chromatography in an octyl-Sepharose column (Pharmacia Biotech, Uppsala, Sweden). The tGPI-mucins were then eluted with a propan-1-ol gradient (5–60%). The criteria of purity and possible contamination with bacterial glycolipids have been determined previously [6
, 8
].
In vivo priming procedure
The in vivo priming of animals was carried out by immunizing animals with FCA, as described previously [19
]. In some experiments, MBSA was added to the FCA to allow the lymphocyte-activation studies described below. Similar in vivo priming was obtained in the absence or presence of that antigen (unpublished results), and in vivo priming experiments were performed in the absence of MBSA. Briefly, animals were given two 50 µl injections of FCA with or without MBSA, i.e., in the shaved abdomen of sedated mice. Animals were then used for the pleurisy experiments described below, 6–7 days after the immunization.
Other experiments were designed to prime animals with systemic IL-12 treatment. Recombinant murine IL-12 was kindly provided by the Genetics Institutes and was used at a concentration of 0.5 µg/animal intraperitoneally (i.p.), 48 and 24 h before the pleural injection of tGPI-mucins.
Pleurisy model
Animals were anesthetized with ether and inflammatory stimuli—tGPI-mucins (10–1250 ng/site), LPS (10–1250 ng/site), chemokines (100 ng/site), or PBS control—given intrapleurally (i.pl.; 100 µl/pleural cavity). Because the molecular weight of LPS is highly variable, and there is no international unit for tGPI-mucins, experiments comparing LPS and tGPI-mucins were performed using a weight basis. Animals were killed at various times (4, 24, 48, or 72 h) after the i.pl. injection of the stimuli, and the cells present in the pleural cavity were harvested by injecting 2 ml PBS. Total cell counts were performed in a modified Neubauer chamber using Turk’s stain, and differential cell counts were performed on cytospin preparations stained with May-Grumwald-Giemsa using standard morphologic criteria to identify cell types. The results are presented as the number of cells per cavity.
Depletion of CD4+ and CD8+ cells
For the depletion of CD4+ lymphocytes, animals were pretreated 4 days after priming with the monoclonal antibodies (mAb) GK1–5 (100 µg/mouse, i.p.) and YTA (200 µg/mouse twice, 3 days apart, i.p.). One day after the last administration of YTA, tGPI-mucins or PBS were administered into the pleural cavity. CD8+ lymphocyte depletion was carried out by pretreating animals with mAb 2–43 (100 µg/mouse, i.p.) 4 days after priming. The depletion of CD4 or CD8 was confirmed by performing fluorescein-activated cell sorter (FACS) analysis (B&D FACScan) of the lymphocyte population in blood and spleen using anti-CD4 or anti-CD8 fluorescein isothiocyanate (FITC)-labeled antibodies (Sigma Chemical Co.).
Ex vivo lymphocyte culture
Spleens were collected aseptically from naïve animals, and animals immunized with FCA/MBSA and teased into single-cell suspension. A pool of spleens from three animals in each group was used for each experiment. Red blood cells were then removed by spinning the cell suspension (15 min, 800 g) over a Ficoll-Paque gradient (Pharmacia-Biotech; 1.077). Cells obtained at the top of the gradient were collected, washed thrice, and resuspended in a final solution of 3 x 106 cells/ml in RPMI containing 10% fetal bovine serum (FBS). Cell suspension (500 µl) was added to each well of 24-well plates and incubated with buffer, MBSA (1–100 µg/ml), or Con A (5 µg/ml). Forty-eight hours later, samples were collected and stored at -70°C until the measurement of IL-4 or IFN-, using a sandwich enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from Pharmingen (San Diego, CA) and according to the protocol of the supplier.
Flow cytometric analysis
Pleural cavity leukocytes were obtained as described above from naïve or primed animals prior to and 48 h after challenge with PBS or tGPI-mucins. Cells were resuspended in PBS (106/ml) containing 0.25% BSA and 0.5 µg/ml FC Block (Pharmingen) and were incubated with the relevant FITC-labeled antibody (CD4, CD8, MAC-3, and Neut; Pharmingen) for 30 min on ice-cold water. The cells were washed twice and analyzed on a B&D FACScan.
Macrophage culture
Naïve C57/BL6 mice were inoculated i.p. with 2 ml 3% thioglycollate, and 4 days later, the elicited peritoneal exudates cells were harvested in cold, serum-free Dulbecco’s modified Eagle’s medium (DMEM). The cells obtained were washed twice in DMEM supplemented with 40 µg/ml gentamicin and 5% heat-inactivated fetal calf serum (complete DMEM) and were resuspended in a final concentration of 2 x 106 cells/ml. Cells (100 µl) were dispensed in 96-well culture plates, and macrophages were allowed to adhere at 37°C and 5% CO2 for 3 h. The plates were washed twice with 200 µl DMEM to remove cells in suspension and incubated in complete DMEM with or without IFN- and/or tGPI-mucins for 48 h at 37°C and 5% CO2. Aliquots of the culture medium were collected at 48 h for MCP-1 measurements as described below.
MCP-1 ELISA
Pleural cavity washes were obtained prior to and 3, 6, 12, and 24 h after challenge with tGPI-mucins or PBS in primed animals. Pleural cavity and cell-culture samples were then centrifuged (500 g for 10 min), and supernatants were assayed in an ELISA set-up using commercially available antibodies (Pharmingen) and according to the instructions provided by the supplier. Briefly, ELISA plates (Nunc Immunosorb, Naperville, IL) were coated with 4 µg/ml purified antibody, incubated overnight, and blocked, and samples or standard were added. These were left overnight, and biotinylated, anti-MCP-1 antibody (1 µg/ml) was added. The reaction was developed using streptavidin-coupled to horseradish peroxidase (HRP; 1:4000 dilution) and OPD (0.4 mg/ml) in H2O2 as a substrate. Sensitivity of the assay was 16 pg/ml.
Immunohistochemistry
Cytospins (Cytospin III, Shandon, 400 rpm, 3 min) were prepared from pleural cavity cells obtained 12 h after tGPI anchor or PBS challenge and fixed in cold (-20°C) acetone for 10 min. Anti-MCP-1 rabbit polyclonal antibody (1/200) or normal rabbit serum was added to the cells and incubated overnight at 4°C. The slides were washed thrice in PBS, and the reaction was developed using the Dako LSAB2 HRP System (Dako, Denmark) and 3,3'-diaminobenzidine as enzyme substrate.
Statistical analysis
Experiments were analyzed by using two-way analysis of variance (ANOVA) on normally distributed data. P values were assigned using the Newman-Keuls procedure, and values of P < 0.05 were considered statistically significant. Percentage inhibition was calculated subtracting background values. Results are presented as the mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[6
, 7
]. For example, the production of NO or IL-12 is elevated markedly after activation with tGPI-mucins in macrophages primed with IFN- [6
, 7
]. To investigate whether in vivo priming would lead to a response after activation with tGPI-mucins, animals were primed with a schedule (MBSA/FCA priming), shown previously to induce the production of IL-12 and IFN- in vivo [19
, 21
]. In these animals, there was a marked, spontaneous production of IFN-, but not IL-4, by splenocytes (Table 1
). Moreover, IFN- production enhanced significantly after activation with a specific antigen (Table 1)
but not with an unrelated antigen (unpublished results) [19
].
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for the priming response by using primed animals that were deficient in IFN- or by priming animals with rIL-12, an IFN--inducing cytokine. As shown in Figure 4A
, tGPI-mucins induced no recruitment of leukocytes in MBSA/FCA-primed IFN--/- mice when compared with primed wild-type animals. Moreover, the priming response could be mimicked by rIL-12 treatment of wild-type animals (Fig. 4B)
in a schedule shown previously to induce significant IFN- production in vivo [22
]. Overall, the data above suggest that the priming response is mediated by IFN- production and requires CD4+ and CD8+ cells.
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had no effect on the recruitment of leukocytes observed after injection of tGPI-mucins (Fig. 5A
). In contrast, the anti-RANTES and anti-MIP-1 antibodies effectively inhibited the inflammatory infiltrate induced by 100 ng/cavity of RANTES or MIP-1 by 80 and 55%, respectively (unpublished results). In agreement with the lack of effect of anti-MIP-1 antibodies, tGPI-mucins induced significant leukocyte recruitment in MIP-1-/- animals (PBS, 2.9±0.1; tGPI-mucins, 7.2±0.4x106 total leukocytes/cavity; n=6; P<0.05). The injection of tGPI-mucins into the pleural cavity of CXCR2-/--primed mice was accompanied by a significant accumulation of mononuclear cells (Fig. 5B)
. However, virtually no eosinophils or neutrophils were found after injection of tGPI-mucins when compared with wild-type animals (Fig. 5B)
.
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, pretreatment of animals with an anti-MCP-1 antibody was accompanied by complete inhibition of the inflammatory infiltrate seen 24 h after injection of tGPI-mucins (Fig. 6A
). Moreover, the injection of tGPI-mucins into primed mice induced a significant increase in pleural wash levels of MCP-1, which was detected first at 12 h and maximal 24 h after challenge (Fig. 6B)
. Inasmuch as MCP-1 was produced in vivo and appeared to play a major role in mediating the recruitment of leukocytes into the pleural cavity of tGPI-mucins-injected mice, a series of experiments was carried out to determine the cell type responsible for MCP-1 production. In pleural cavity cells stained with an anti-MCP-1 antibody, immunoreactivity was restricted virtually to macrophages (unpublished results; see http://www.icb.ufmg.br/
impar/ilust_1.htm). Of note, most macrophages were MCP-1-positive, but there were macrophages that were not stained with the antibody. Neutrophils and lymphocytes did not stain with anti-MCP-1 antibody after injection of tGPI-mucins (unpublished results). We then investigated the ability of murine macrophages to respond to stimulation with tGPI-mucins. Activation of macrophages in vitro with tGPI-mucins or IFN- alone induced some MCP-1 production (Fig. 7A
). However, when IFN--primed macrophages were activated with tGPI-mucins, these stimuli synergized to induce a marked production of MCP-1 (Fig. 7A)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and NO). The studies mentioned above suggest that T. cruzi-derived tGPI-mucins, which are quantitatively dominant molecules present in the host/parasite interface, may be important determinants of a host/parasite relationship and disease outcome during this protozoan infection. Here, we have sought evidence for a role of tGPI-mucins in mediating leukocyte recruitment in vivo.
The injection of tGPI-mucins in naïve animals induced no inflammatory response, whereas injection of bacterial LPS induced a dose- and time-dependent recruitment of leukocytes. These in vivo results agree with the little effect of tGPI-mucins when used to activate unprimed macrophages in vitro [6
, 7
]. In vitro, macrophages primed with IFN- produce higher levels of inflammatory cytokines and NO in response to tGPI-mucins [6
7
8
9
]. Thus, we tested whether tGPI-mucins would also induce an inflammatory response in vivo after priming the whole animal. To this end, we used a priming schedule shown previously to induce high levels of IFN- in vivo (see Table 1
, and refs. [19
, 21
]). Under priming conditions in vivo, tGPI-mucins induced a significant dose- and time-dependent leukocyte recruitment. Of note, polymorphonuclear leukocytes migrated early, whereas mononuclear cells and especially MAC-3-expressing macrophages predominated later in the inflammatory reaction. Priming was not observed when animals were depleted of CD4+ or CD8+ cells and did not occur in IFN--/- mice. Moreover, priming could be mimicked by the administration of rIL-12 to wild-type mice in doses shown previously to elevate the in vivo levels of IFN- [22
]. It is interesting that the production of IFN- by immunization with MBSA in FCA has been demonstrated to depend on the production of IL-12 [21
]. Altogether, our data suggest that the priming of animals with FCA relies on activation of CD4+ and/or CD8+ cells, which ultimately induces the production of IFN- that drives the priming to tGPI-mucins-induced inflammation. As tGPI-mucins may induce IL-12 even in the absence of IFN-, albeit at small quantities [6
, 9
], it remains to be determined whether live T. cruzi trypomastigotes or tGPI-mucins themselves could provide a T-cell-independent source of IFN- and tissue priming for leukocyte recruitment. It is of note that IFN--deficient animals infected with T. cruzi have little chemokine expression and inflammation but a large number of parasites in the affected tissues [13
].
LPS and other pathogen-associated molecular patterns depend on TLR signaling pathways to activate the innate immune system [26
27
28
]. To investigate whether tGPI-mucins functioned via activation of TLR4, akin to LPS [29
], we used C3H/HeJ mice, which have dysfunctional TLR4 [23
, 24
]. Our results demonstrate clearly that whereas LPS-induced inflammation does not occur in this mouse strain, the leukocyte accumulation in response to injection of tGPI-mucins was still present (see Fig. 8
). These results are in agreement with our previous in vitro experiments demonstrating that tGPI-mucins, but not LPS, activate C3H/HeJ-derived macrophages to produce IL-12, TNF-, and NO [6
, 7
]. Again, in agreement with our in vitro observations using inflammatory or bone marrow-derived macrophages, it is interesting that tGPI-mucins failed to induce MCP-1 production in vitro and an inflammatory response in TLR2-deficient animals [30
]. In support of our previous studies [8
], tGPI was the part of the molecule responsible for the activation of the TRL-2 receptors on macrophages [30
]. Thus, this receptor is important not only for cytokine and NO production in vitro but also for the ability of tGPI-mucins to induce leukocyte recruitment in vivo. To the best of our knowledge, this is the first study demonstrating an in vivo role for TLR2 in protozoan-induced inflammation.
Chemokines are low molecular weight cytokines whose major biological function is to mediate leukocyte trafficking [15
, 31
]. A role for chemokines in the pathophysiology of several infectious and noninfectious diseases has been suggested but demonstrated only for HIV-1 and Plasmodium vivax infection [31
]. We have shown that chemokines were expressed during acute and chronic experimental Chagas in mice, and there appeared to be a correlation between chemokine expression and leukocyte subset infiltration [13
, 14
]. Of note, the chemokines MIP-1, RANTES, and MCP-1 were greatly expressed during infection and could activate infected macrophages to produce NO and kill T. cruzi [12
]. Using antichemokine antibodies or gene-deficient animals, we assessed the role of chemokines in mediating the inflammatory infiltrate following injection of tGPI-mucins in vivo. We show that leukocyte migration at 24 h is largely dependent on the local production of MCP-1 but not MIP-1 or RANTES. In addition, the migration of neutrophils but not mononuclear cells at that time point was largely dependent on the activation of CXCR-2 receptors. This is in agreement with the important role of this receptor for neutrophil recruitment in several models of inflammation in vivo [19
, 31
]. MCP-1 was detected first 12 h after tGPI-mucins injection, and immunocytochemistry showed that most of immunoreactivity was expressed by macrophages. In support of the important role of macrophages in the production of MCP-1 in vivo, in vitro activation of IFN--primed macrophages with tGPI-mucins induced the production of considerable amounts of MCP-1. These results are in agreement with other studies showing the importance of this chemokine for monocytic cells recruitment in vivo [32
] and the ability of LPS-activated macrophages to induce MCP-1 production in vitro [33
].
The activation of TLR2 receptors by T. cruzi trypomastigote-derived tGPI-mucins may play an important role in the development of a specific immune response and host resistance to this parasite via the facilitation of the production of proinflammatory cytokines and NO. Several of these activities of tGPI-mucins are dependent on the priming of macrophages by IFN-. We now show the ability of tGPI-mucins, in conjunction with IFN-, to induce in vivo chemokine production and inflammation in a TLR2-dependent manner. Together, our results suggest that T. cruzi-derived tGPI-mucins may drive tissue inflammation and bear a significant role in the pathogenesis of Chagas disease. Future studies using mice that lack the TLR2 receptor may provide further information about the in vivo role of tGPI-mucins in the immunopathogenesis of Chagas disease.
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ACKNOWLEDGEMENTS |
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Received October 17, 2001; revised December 6, 2001; accepted December 21, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-primed macrophages J. Immunol. 159,6131-6139[Abstract]
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as important determinants of chemokine mRNA expression during infection with Trypanosoma cruzi Microbes Infect. 2,851-866[Medline]
T lymphocytes in lipopolysaccharide-induced eosinophil accumulation into the mouse pleural cavity J. Immunol. 159,853-860[Abstract]
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production J. Immunol. 153,1697-1706[Abstract]
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