INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Interleukin-10 (IL-10) is a pleiotropic immunomodulatory cytokine, produced by a wide variety of cells, including activated TH2 cells, monocytes and macrophages, B cells, thymocytes, and keratinocytes (19, 21, 23, 26, 46, 64). IL-10 plays a pivotal role in the establishment and maintenance of a class of immune response by suppressing TH1-dependent cell-mediated immunity and augmenting TH2-dependent immune responses (22, 49). Through the prevention of macrophage activation, as well as via direct interaction, IL-10 has been shown to prevent antigen (Ag)-specific T-cell stimulation, proliferation, and cytokine production indirectly by reducing the Ag-presenting ability of monocytes (18, 22). This effect is associated with the downregulation of major histocompatability complex (MHC) class II molecules (10), and costimulatory molecules such as B 7.1, B 7.2, and ICAM-1 (10, 66). IL-10 also potently suppresses many effector functions of monocytes and macrophages, including the release of cytokines such as gamma interferon, tumor necrosis factor alpha (TNF-), IL-1, IL-6, IL-12, C-X-C, and C-C chemokines (1, 6, 7, 9, 35, 36, 47, 58) and the generation of nitric oxide (NO) (24).

While cell-mediated immunity is a critical prerequisite for effective clearance of a microbial invader, modulation of the inflammatory response is equally important in order to ensure preservation of immune homeostasis. As a potential immunomodulator, IL-10 favors the attenuation of host defense mechanisms against pathogenic invasion and facilitates the progression of the disease. In support of this premise, infection with Mycobacterium avium and Klebsiella pneumoniae induced production of IL-10 and administration of anti-IL-10 antibody developed resistance to infection in mice (4, 16, 29). Furthermore, exaggerated expression of IL-10 in patients with leprosy is associated with persistent and chronic infection (60). Recently, it has been reported that IL-10 inhibits the intracellular killing of Leishmania major (65), and human IL-10 transgenic mice that released elevated levels of IL-10 developed a much more progressive lesion and parasite burden than nontransgenic control mice when both were infected with L. major (30). A high level of splenic IL-10 expression was observed in the murine model of visceral leishmaniasis, which in turn contributed to the suppression of splenic T-cell function and was associated with multiplication of visceral parasites (44, 67). However, the intracellular signaling mechanism encompassing IL-10-mediated attenuation of the host response in visceral leishmaniasis has not been investigated.

Several studies have implicated protein kinase C (PKC) in the control of intracellular microbial replication. In this context, the leishmanial parasite has gained a great deal of attention because it impairs PKC-dependent signaling in infected macrophages (8, 17, 55). Inhibition of PKC enhances intracellular multiplication of L. donovani (16, 48, 51). Such observations have led to the proposition that PKC might be considered as a host resistance determinant against leishmanial infection (16, 48). In the present study we sought to characterize the role of IL-10 in the alteration of signal transduction events in murine visceral leishmaniasis. We have previously observed that infection with Leishmania donovani selectively inhibits the activity and expression of Ca⁺²-dependent classical PKC (4a). Our findings suggest that such impairment might be facilitated by the overproduction of IL-10 in macrophages under parasitic stress. The activity of PKC in infected macrophages was significantly restored by pretreatment with neutralizing anti-IL-10 monoclonal antibody (MAb). Moreover, endogenous release of IL-10 down-regulated the host-mediated oxidative and inflammatory responses during parasitic challenge, which in turn favored the survival of the protozoan within the host.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Animals and parasites. BALB/c mice were purchased from the National Centre for Laboratory Animal Sciences, India. For each experiment, 8 to 10 mice (4 to 6 weeks old) were used, irrespective of sex.

Preparation of peritoneal macrophages. Mouse macrophages were isolated by peritoneal lavage 48 h after intraperitoneal injection of 1.0 ml of sterile 4% thioglycolate broth (Difco Laboratories, Detroit, Mich.). The peritoneal macrophages were collected by infusing the peritoneal cavity with ice-cold sterile phosphate-buffered saline. Cells were cultured as described by Fahey et al. (20).

Preparation of cell lysate. The adherent cell population was scraped and centrifuged at 400 × g for 15 min at 4°C. The cells were then resuspended in ice-cold extraction buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM EGTA, antiprotease mixture, and 50 mM -mercaptoethanol. The antiprotease mixture consisted of 0.33 mM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 0.35 mM antipain, 0.24mg of chymostatin per ml, 0.35 mM pepstatin, and 4.8 TIU of aprotinin per ml (12, 42). The macrophage-containing suspension was sonicated at 4°C and centrifuged at 4,250 × g for 10 min at 4°C, and the supernatant was used for the experiments.

Cytokine analysis by sandwich ELISA. The level of mouse IL-10 in the conditioned medium of macrophage culture was measured using the enzyme-linked immunosorbent assay (ELISA) kit (Quantikine M; R&D System, Minneapolis, Minn.). The minimum detectable dose of mouse IL-10 was found to be typically less than 4.0 pg/ml. TNF- levels were quantified using a mouse TNF- ELISA kit (Factor-Test-X; R&D System). The detection limit of this assay was determined to be 15.0 pg/ml. The assay was performed by following the detailed instructions of the manufacturer.

Treatment with rIL-10 and neutralizing anti-IL-10 MAb. Macrophages were cultured overnight and then treated with 10 ng of mouse recombinant IL-10 (rIL-10) (R&D System) per ml, and the cells were cultured in fresh, complete RPMI-1640 for various periods according to the experimental protocol. To confirm the effects of rIL-10, simultaneous experiments were performed by preincubating parasitized macrophages with neutralizing rat anti-mouse IL-10 MAb and isotype-matched control rat immunoglobin G (IgG), both from R&D System, at a dose of 4 µg/ml for 30 min at room temperature.

Endogenous protein phosphorylation. Macrophages were sonicated in EGTA-containing buffer and centrifuged at 4,250 × g for 10 min at 4°C. The supernatant was used as the source of both endogenous substrates and enzymes. The reaction mixture contained 20 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 0.25 mM EGTA, 1 mM NaF, 0.1 mM sodium vanadate, 20 µg of phosphatidyl serine (PS) per ml, 2 µg of diglyceride (DG) per ml, 50 µM [-³²P]ATP, and 100 µg of endogenous protein, in the presence or absence of 0.6 mM CaCl₂, in a total volume of 50 µl. Incubation was carried out for 30 min at 30°C (12, 42). The reaction was stopped by adding Laemmli buffer; the mixture was then boiled for 5 min, separated by 5 to 20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to autoradiography.

PKC assay. PKC activity was assayed in a PS/DG- and Ca/PS/DG-dependent manner by measuring the incorporation of -³²P (BARC, Mumbai, India) into histone type III S (Sigma Chemical Co., St. Louis, Mo.), as described by Das et al. (12) and Majumdar et al. (42). PKC activity study was extended in the presence and absence of pseudosubstrate (Pss) peptide to inhibit a specific isoform of PKC. Pss peptide [Ala²⁵] (19 to 36 residues) for -PKC, having the sequence RFARKGALRQKNVHEVKN, was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Pss peptide [Ala¹¹⁹] (113 to 130 residues) for -PKC, having the sequence SIYRRGARRWRKLYRANG, was a generous gift from A. Bannerjee, Cleveland Clinic Foundation, Cleveland, Ohio. These Pss peptides are derived from the N terminus of PKC (33).

Electrophoresis and immunoblotting. Whole-cell sonicate was allowed to centrifuge at 4,250 × g for 10 min at 4°C to remove the nuclear fraction. The supernatant was separated on an SDS-10% PAGE and transferred to nitrocellulose membrane. The membrane was blocked overnight with 3% bovine serum albumin in Tris-saline buffer (pH 7.5), and immunoblotting was achieved as described by Das et al. (12) and Majumdar et al. (42). Immunoreactive bands were visualized using nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate as a chromogenic substrate for alkaline phosphatase.

Assay for CaM kinase II activity. The activity of CaM kinase II, a Ca⁺²/calmodulin-dependent protein kinase type II was assayed by measuring the transfer of -³²P from [-³²P]ATP into the synthetic peptide substrate, syntide-2. Syntide-2 has the sequence Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys. The reaction mixture contained an aliquot equivalent to 5 × 10⁴ cells and was incubated in 50 µl of reaction mixture consisting of concentrations of 20 µM syntide-2, 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 50 µM (1 µCi) [-³²P]ATP in the presence or absence of 5 µM bovine calmodulin, and 0.5 mM CaCl₂. The reaction mixture was incubated at 25°C for 5 min. CaM kinase II activity study was extended in the presence and absence of 30 µM W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide], a potent calmodulin antagonist. The syntide-2, calmodulin, and W-7 were provided by Maitrayee Dasgupta (Department of Biochemistry, Calcutta University, Calcutta, India).

Uptake and intracellular multiplication of L. donovani. Macrophages were cultured on cover slips (1 × 10⁶/ml) with neutralizing anti-IL-10 MAb or isotype-matched control rat IgG (cAb) for 1 h and then challenged with L. donovani promastigotes at a macrophage-to-parasite ratio of 1:10. To study the uptake of parasites, after 4 h of promastigote challenge the noningested parasites were removed by extensive washing with complete conditioned RPMI 1640 and subjected to cell fixation, followed by Giemsa staining (61). To investigate the intracellular multiplication of L. donovani, the washed macrophages were cultured for another 68 h in fresh RPMI medium with neutralizing anti-IL-10 MAb or isotype-matched control rat IgG (cAb) and subjected to Giemsa staining. The percentages of infected macrophages after 4 h and 72 h of L. donovani challenge were 90 to 92% and 88 to 90%, respectively.

Superoxide anion generation. Superoxide anion (O₂) generation was monitored using the superoxide dismutase inhibitable cytochrome c reduction method (3, 50). Briefly, an aliquot of 2 × 10⁶ cells was immediately resuspended in 10 mM HEPES buffer and O₂ generation was measured spectrophotometrically in the presence of 10⁷ M N-formylmethionyl leucyl phenylalanine (fMLP) at 550 nm.

Nitrite assay. The generation of nitrite in the conditioned medium of macrophage culture was assayed by the Griess reaction (28) using a Nitric Oxide Colorimetric Assay Kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). In brief, nitrate present in the sample was reduced to nitrite by the addition of NADPH in the presence of the enzyme nitrate reductase. For the assay, macrophages were cultured in a 24-well tissue culture plate (Falcon) at a concentration of 10⁶ cells per ml. Cell-free culture supernatant was collected, and the nitrite level was estimated according to the manufacturer's instruction.

Densitometric analysis. Autoradiographs of endogenous protein phosphorylation and immunoblots were analyzed using a model GS-700 Imaging Densitometer and Molecular Analyst version 1.5 software (Bio-Rad Laboratories, Hercules, Calif.).

Statistical analysis. Results were expressed as the mean plus or minus the standard deviation (SD) for individual sets of experiments. Each experiment was performed four to five times, and representative data from each set of these experiments were presented in the manuscript. A one or two-tailed t test for significance was performed as applicable in each case. A P value of less than 0.05 was considered significant.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Release of mIL-10 in response to L. donovani infection. We investigated the production of murine IL-10 (mIL-10) by macrophages in primary in vitro culture. Culture supernatant was collected at different periods during incubation, i.e., 4, 12, 18, 24, and 48 h after infection, with or without L. donovani. The release of IL-10 by the uninfected control macrophages was very low, whereas it was dramatically enhanced in parasitized macrophages. After 24 h of infection there was a 16.5-fold increase in the IL-10 level in the culture supernatant compared to that in the uninfected control macrophages (Fig. 1). The level of secretion in the supernatant persisted even after 48 h of infection. Macrophages were also treated with 10 ng of lipopolysaccharide (LPS) per ml, since LPS is a known inducer of IL-10 secretion, to compare the level of mIL-10 release in response to L. donovani infection (Fig. 1).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Release of mIL-10 by BALB/c peritoneal macrophages with or without L. donovani promastigotes. Macrophages (m) were cultured in 96-well plates (2 × 106 cells/ml) and treated with parasites (1:10 ratio) for 4 h. Noningested parasites were removed by washing, and the parasitized macrophages were further cultured with complete RPMI-1640 medium for the periods shown in the figure. IL-10 levels in the culture supernatant were measured by sandwich ELISA. Results are expressed as the mean ± SD of data from quadruplicate experiments. **, statistically significant (P < 0.01) induction of mIL-10 release. *, statistically significant (P < 0.02) induction of mIL-10 release.

Effect of isotype specific pseudosubstrate on PKC activity in response to rIL-10 treatment. To characterize the alteration in the signal transduction profile due to IL-10 overproduction by parasitized macrophages, we assayed the activity of PKC in response to treatment with 10 ng of rIL-10 per ml. We investigated the activity of both Ca²⁺-dependent and -independent PKC isoforms. In control macrophage, considerable PKC activity was observed in the presence of Ca²⁺/PS/DG (Fig. 2A), whereas in rIL-10-treated macrophages Ca²⁺/PS/DG-dependent PKC activity was reduced by 61.73% (Fig. 2A) compared to that in control macrophages (considered as 100%). Infection with L. donovani also inhibited Ca²⁺/PS/DG-dependent PKC activity by 67.63%. It is interesting that the activity was restored by anti-IL-10 neutralizing MAb, whereas the isotype-matched control IgG (cAb) had no effect (Fig. 2A). On the other hand, Ca²⁺-independent, but PS/DG-dependent, PKC activity remained almost unaltered in rIL-10-treated macrophages, although leishmanial infection enhanced the PKC activity of these macrophages (Fig. 2B).

View larger version (51K): [in this window] [in a new window]   FIG. 2.   (A) Effect of -Pss on PKC activity. Macrophages were cultured overnight and incubated with L. donovani promastigotes for 4 h. Noningested parasites were removed by washing with complete RPMI 1640 and further incubated for 20 h. Cells were sonicated in EGTA-containing buffer (see Materials and Methods), and the cell sonicate was used as the source of endogenous kinases. PKC activity was assayed in the presence of Ca, PS, and DG, with or without 5 µM -Pss. Data are expressed as the percentages of activity of uninfected macrophages in the presence of Ca, PS, and DG, where 100% value is 109.77 pmol/min1 mg1 of protein. Each result represents the mean ± SD of data from four independent experiments. *, statistically significant inhibition (P < 0.05) of PKC activity in the absence of -Pss. **, statistically significant inhibition (P < 0.05) of PKC activity in the presence of -Pss. (B) Effect of -Pss on PKC activity. Activity of PKC was assayed in the presence of PS and DG, with or without 5 µM -Pss. Data are expressed as the percentages of activity of the uninfected macrophages in the presence of PS and DG, where 100% value is 58.36 pmol min1 mg1 of protein. Each result represents the mean ± SD of data from four independent experiments. *, statistically significant inhibition (P < 0.05) of PKC activity in the presence of -Pss. **, statistically significant inhibition (P < 0.03) of PKC activity in the presence of -Pss. ***, statistically significant activation (P < 0.05) of PKC activity in the absence of -Pss.

Endogenous protein phosphorylation. The result reported above prompted us to study the phosphorylation status of macrophage-derived proteins. In the control macrophage, significant phosphorylation of 67-, 54-, 47-, and 36-kDa proteins was observed in a PKC-dependent manner in the presence of the activators Ca²⁺, PS, and DG (Fig. 3A, lane 3). Infection with AG-83 inhibited the Ca²⁺/PS/DG-dependent protein phosphorylation (Fig. 3A, lane 6). Densitometric scanning analysis revealed that the inhibition of Ca²⁺/PS/DG-dependent phosphorylation of 67-, 54-, 47-, and 36-kDa proteins was 46.24, 83.65, 38.18, and 84.99%, respectively, compared to that in the uninfected macrophages (Fig. 3B). It is interesting that phosphorylation of 67-, 54-, and 47-kDa proteins was restored in infected macrophages preincubated with neutralizing anti-IL-10 MAb (Fig. 3A, lane 9).

View larger version (65K): [in this window] [in a new window]   FIG. 3.   (A) Endogenous protein phosphorylation of BALB/c peritoneal macrophages. Cells were cultured with or without L. donovani promastigotes as described in Fig. 2A. Cell sonicate was used as the source of both endogenous substrates and kinases. Phosphorylation reaction was carried out with MgCl2, PS, DG, and [-32P]ATP in the presence (+) and absence () of CaCl2. Proteins were solubilized in Laemmli buffer and subjected to SDS-5 to 20% gradient PAGE followed by autoradiography. (B) Densitometric scanning analysis for the relative phosphorylation of 67-, 54-, 47-, and 36-kDa proteins, considering 100% as the phosphorylation of each individual protein in control macrophage. The autoradiogram is from one of three independent experiments, all of which yielded similar results.

Expression of - and -PKC isoforms. After the above observations, it was necessary to investigate whether selective impairment of PKC isoforms in response to rIL-10 was also reflected at the level of protein expression (described in Materials and Methods). Densitometric scanning analysis revealed that there was a 39.35% inhibition of -II PKC expression (Fig. 4B). However, no significant change in -I PKC (Fig. 4A) and -PKC (Fig. 5) expression was detected in response to rIL-10 challenge.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Expression of -I and -II PKC isotypes. Cells were cultured with or without L. donovani promastigotes as described in Fig. 2A. Immunoblotting was achieved using specific (A) -I PKC (lanes 1 and 2) and (B) -II PKC (lanes 1 and 2) antibodies. Lanes 1 and 2 in both panels represent immunoreactive bands for the control and for rIL-10-treated macrophages, respectively. Densitometric scanning was done for each immunoreactive band. Data presented are from one of three independent experiments, which yielded similar results.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   Expression of -PKC isotype. Cells were cultured with or without L. donovani promastigotes as described in Fig. 2A. Immunoblotting was achieved using specific -PKC antibody (see Materials and Methods). Lanes 1 and 2 represent immunoreactive bands for the control and for rIL-10-treated macrophages, respectively. Densitometric scanning was done for each immunoreactive band. Data presented are from one of three independent experiments, which yielded similar results.

CaM kinase activity in response to rIL-10 treatment. The inhibition of the Ca²⁺-dependent PKC isoform prompted us to study the activity of another Ca²⁺-dependent protein kinase, CaM kinase II, a Ca²⁺/calmodulin-dependent protein kinase. In uninfected control macrophages, significant CaM kinase II activity was observed which was almost completely inhibited with W-7, a CaM kinase II-specific inhibitor (Fig. 6). Treatment with rIL-10 inhibited CaM kinase II activity; the activity was reduced by 48.32% compared to that in control macrophages (Fig. 6). Inhibition of CaM kinase II was also observed in L. donovani-infected macrophage, in which the activity was reduced by 69.28% (Fig. 6). Preincubation of the L. donovani-infected macrophages with anti-IL-10 MAb caused 92.38% of the CaM kinase II activity to be recovered (Fig. 6). We could not identify any significant recovery as the result of the prior treatment of the infected macrophage with isotype-matched cAb (Fig. 6).

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Effect of L. donovani promastigotes and rIL-10 treatment on CaM kinase II activity of BALB/c peritoneal macrophages. CaM kinase II activity was assayed in the presence of Ca/CaM or, Ca/CaM/W-7. Cells were cultured with or without L. donovani promastigotes as described in Fig. 2A. Cell sonicate was used as the source of endogenous kinases. Each result represents the mean ± SD of data from four independent experiments. *, statistically significant (P < 0.02) inhibition of CaM kinase II activity. **, statistically significant (P < 0.05) inhibition of CaM kinase II activity. ***, statistically significant (P < 0.05) recovery of CaM kinase II activity.

Parasite uptake and multiplication. Our observations showed several biochemical changes in leishmaniasis in response to endogenous secretion of mIL-10. Eventually, it appeared quite necessary to understand whether the endogenous release of IL-10 exerts any role in the uptake and intracellular multiplication of L. donovani. To monitor parasite uptake, parasite burden was observed after 4 h of infection. There was no significant change in parasite entry when the infected macrophages were preincubated with either anti-IL-10 MAb or isotype-matched cAb (Fig. 7). The relative profile of parasite burden was dramatically changed when we allowed the infection to proceed for 72 h. It is interesting that pretreatment with anti-IL-10 MAb reduced the parasite burden by 36.35% in comparison to that in the control-infected macrophages (Fig. 7). This was probably due to the neutralization of endogenous mIL-10 secretion by the parasitized macrophages. Preincubation with isotype-matched cAb did not confer any remarkable effect on parasite multiplication (Fig. 7).

View larger version (26K): [in this window] [in a new window]   FIG. 7.   Effect of rIL-10 on L. donovani promastigote uptake and intracellular multiplication within BALB/c peritoneal macrophages. Treatment of macrophages with L. donovani promastigotes, neutralizing anti-IL-10 MAb, and isotype-matched cAb was done as described in Materials and Methods. Results are presented as the mean ± SD of data from five replicate experiments. *, statistically significant (P < 0.03) enhancement of L. donovani multiplication at 72 h after infection compared to at 4 h after infection. **, statistically significant (P < 0.05) inhibition of L. donovani multiplication in response to anti-IL-10 MAb with regard to untreated infected macrophages.

Generation of nitrite. Next, we ascertained whether the enhanced parasite burden, in response to rIL-10, accounts for the immunosuppression that ensures the intracellular parasite survival via macrophage deactivation. It has been documented that NO inhibits the growth and multiplication of a diverse array of microorganisms (68). In our study, we estimated the level of nitrite in the culture supernatant, which is an established indirect method to measure the release of NO. We observed that infection with L. donovani almost completely inhibited the generation of nitrite, which was significantly recovered by the incubation with anti-IL-10 MAb prior to leishmanial challenge (Fig. 8). To understand the role of rIL-10 in this context, we observed nitrite generation in rIL-10-treated macrophages. In the presence of rIL-10, the production of nitrite was inhibited by 3.6-fold compared to that in control macrophages (Fig. 8).

View larger version (25K): [in this window] [in a new window]   FIG. 8.   Effect of L. donovani promastigotes and rIL-10 treatment on the generation of nitrite by BALB/c peritoneal macrophages. Cells were cultured in a 24-well plate (106 cells/ml) with LPS (1 µg/ml). Macrophages were treated with L. donovani or rIL-10 for 48 h. The level of nitrite in the culture supernatant was evaluated as described in Materials and Methods. Results are presented as the mean ± SD of data from four replicate experiments. ND, level of nitrite is not detectable under respective treatment. *, statistically significant (P < 0.03) inhibition of nitrite generation. **, statistically significant (P < 0.02) inhibition of nitrite generation. ***, statistically significant (P < 0.05) recovery of nitrite generation.

Release of mTNF-. TNF- acts as a triggering signal for NO generation (27). We attempted to study the endogenous release of murine TNF- (mTNF-) in response to L. donovani and rIL-10 treatment. There was an 82.05% inhibition of mTNF- release by rIL-10-treated macrophages in comparison to that in control macrophages (Fig. 9). Inhibition of mTNF- release in L. donovani-infected macrophages was comparable to that in macrophages treated with rIL-10, but the inhibition was only partially recovered (64.93% of that in the control) by preincubation with anti-IL-10 MAb (Fig. 9).

View larger version (31K): [in this window] [in a new window]   FIG. 9.   Release of mTNF- by BALB/c peritoneal macrophages with or without L. donovani promastigotes and rIL-10. Macrophages were cultured as mentioned in Fig. 1 and treated with L. donovani for 24 h. The mTNF- level in the cell culture supernatant was measured by sandwich ELISA. Results are expressed as the mean ± SD of data from quadruplicate experiments. *, statistically significant (P < 0.02) inhibition of mTNF- release. **, statistically significant (P < 0.03) recovery of mTNF- release.

Generation of the O₂ anion. We further attempted to estimate the generation of the O₂ anion, which is considered an important oxidative-defense mechanism adopted by phagocytes against microbial invasion. Normal macrophages stimulated with fMLP produced a considerably higher level of O₂ (Fig. 10). Treatment with either L. donovani or rIL-10 caused substantial inhibition of O₂ production (Fig. 10). The inhibition was 76% and 61.5%, respectively, compared to that in the untreated control. It is interesting that pretreatment with anti-IL-10 MAb partly restored the oxidative response in infected macrophages (Fig. 10). We could not detect any significant effect in O₂ anion generation when the control macrophages were treated with either anti-IL-10 MAb or isotype-matched control IgG (data not shown).

View larger version (26K): [in this window] [in a new window]   FIG. 10.   Superoxide anion O2 generation in BALB/c peritoneal macrophages. Macrophages were cultured for 24 h after respective treatment. The generation of the O2 anion was measured as the superoxide dismutase inhibitable cytochrome c reduction at 550 nm, and the change in optical density was monitored every 30 s. Results are expressed as the mean ± SD of data from four independent experiments and expressed as n moles of O2/2 × 106 cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In the immune system, macrophages confer a pivotal role in both the regulation of homeostatic processes and a broad spectrum of acute and chronic inflammatory diseases. Investigation encompassing the activation-deactivation mechanism of macrophages thus may provide an insight into the role of immunoeffector cells in the pathogenic responses. Several physiologic mediators which act as macrophage deactivators have been identified. It is documented that IL-10 is a potential inhibitor of cytokine synthesis, gene expression, and consequent protein synthesis in macrophages, but the mechanism involved in such deactivation is still unknown.

Northern blot analysis and qualitative PCR of total BALB/c liver RNA indicated that IL-10 mRNA expression was induced in L. donovani-infected mice (44), although the immunobiologic efficacy of transcriptionally induced IL-10 remained to be ascertained. Consistent with this report, we observed that in vitro infection of BALB/c peritoneal macrophages with L. donovani caused significant induction of IL-10 release in the culture supernatant. There was a 16.5-fold increase in release of IL-10 by the parasitized macrophages after 24 h of infection compared to that in uninfected control macrophages (Fig. 1).

The major objective of this study was to characterize the signaling events leading to IL-10-driven macrophage deactivation in visceral leishmaniasis. PKC is considered an important host resistance determinant, and it appeared to be essential for the induction of proinflammatory cytokines (viz. TNF- expression) in macrophages (11, 38). Recently, it has been reported that IL-10 inhibits the inflammatory response in alveolar macrophages by modulating PKC activity and that the activation of PKC by phorbol myristate acetate treatment restores the inflammatory response by augmenting the transcription and translation of TNF- (39). Previously, it was reported by Olivier et al. (55), Giorgione et al. (25), and our group (4a) that L. donovani caused impairment of PKC activity. In this context, we attempted to study whether the elevated level of IL-10 released by infected macrophages is involved in the modulation of PKC activity.

Multiple forms of PKC are well documented in macrophage (34, 57). These forms can be differentiated with respect to their intracellular distribution, cofactor requirement, and substrate specificity (42). We observed both the activity of Ca²⁺-dependent and Ca²⁺-independent PKC isoforms. Treatment with either rIL-10 or L. donovani inhibited the activity of Ca²⁺/PS/DG-dependent PKC activity, and the extent of inhibition was comparable (Fig. 2A). Preincubation with anti-IL-10 MAb successfully recovered Ca²⁺/PS/DG-dependent PKC activity in the parasitized macrophages (Fig. 2A). This observation raised the possibility that endogenous release of IL-10 by the parasitized macrophages might cause impaired Ca²⁺/PS/DG-dependent PKC activity. This finding was in agreement with our endogenous protein phosphorylation study that demonstrated substantial inhibition of 67-, 54-, 47-, and 36-kDa proteins and recovery by the use of neutralizing anti-IL-10 MAb (Fig. 3A). Treatment with rIL-10 did not reveal any significant change of the Ca²⁺-independent activity, but did show changes in PS/DG-dependent PKC activity (Fig. 2B). To understand the involvement of specific PKC isoforms, we studied PKC activity in the presence and absence of Pss inhibitor against Ca²⁺/PS/DG-dependent -PKC and Ca²⁺-independent -PKC. Our study suggested that endogenous release of IL-10 inhibits the activity of -PKC in infected macrophages (Fig. 2A), although Ca²⁺-independent PKC activity remained unaffected (Fig. 2B).

After these observations, we further attempted to study the expression of PKC isoforms. The expression of the Ca²⁺/PS/DG-dependent -II PKC was inhibited by rIL-10 (Fig. 4B) and also by parasitic stress (4a). We could not detect any significant change in the expression of Ca²⁺-independent -PKC in response to rIL-10 (Fig. 5); hence, the immunoblot analysis was at par with our PKC activity study.

It has been reported that the concentration of intracellular Ca²⁺ increases during leishmanial infection (54). Since our observations implicated selective impairment of the Ca²⁺-dependent PKC isoform, we speculated that there might be some dysfunction in Ca²⁺ signaling. We examined the activity of CaM kinase II, another Ca²⁺-dependent protein kinase. In control macrophages, we could detect considerable CaM kinase II activity, and this activity was completely abrogated by preincubation with W-7 (Fig. 6), a well-documented inhibitor of CaM kinase II. Treatment with either rIL-10 or L. donovani inhibited the CaM kinase II activity (Fig. 6). Preincubation with neutralizing anti-IL-10 MAb successfully restored the CaM kinase II activity in L. donovani-infected macrophages (Fig. 6). This observation further strengthened our hypothesis that the high level of endogenous secretion of IL-10 during infection with L. donovani might cause impairment of Ca²⁺-dependent host signal transduction.

To substantiate the defective activation of PKC and CaM kinase II in leishmaniasis in response to IL-10, we made an attempt to assay the generation of the O₂ anion. This was done because the essential prerequisite for O₂ anion generation is the activation of NADPH-oxidase, which is mediated by PKC-dependent phosphorylation events (41, 52). It has been documented that IL-10 inhibits macrophage-mediated bactericidal activity via the inhibition of O₂ generation (6). Previously, we observed impaired O₂ generation in the parasitized macrophages (4a). In our case, treatment with rIL-10 caused a remarkable inhibitory effect on macrophage-mediated O₂ generation (Fig. 10). Moreover, prior incubation with anti-IL-10 MAb could partially restore O₂ production in infected macrophages. This observation was in agreement with the inhibition of Ca²⁺-dependent PKC, as mentioned earlier.

It has been well documented that during pathogenic invasion, the host releases NO, which acts as a potent cytotoxic-cytostatic effector molecule and inhibits the growth and function of a diverse array of infectious agents, including protozoan and helminthic parasites (68). The increase of NO and its role in the control of various intracellular pathogens has been described for leishmaniasis (39), for malaria (63), and for trypanosomal (56), viral (5), and fungal (2) infections. In the control macrophages, we detected a considerable amount of nitrite, which was almost nondetectable in infected macrophages. It is interesting that pretreatment with anti-IL-10 MAb caused a remarkable recovery of nitrite production (Fig. 8), suggesting that endogenous release of IL-10 by parasitized macrophages was involved in the attenuated cytotoxic response via NO. It has been reported that TH1-type proinflammatory cytokine TNF- acts as a triggering signal for NO generation (27) and that PKC is critically involved in the expression of this proinflammatory cytokine (11, 38). To correlate the defective activation of PKC with attenuated nitrite generation, we measured the level of TNF- in response to L. donovani challenge as well as rIL-10 treatment. We observed inhibition of endogenous TNF- release in response to either L. donovani or rIL-10 (Fig. 9). Pretreatment with anti-IL-10 MAb only partially restored the release of TNF- in the parasitized macrophages (Fig. 9). This result raised the possibility of parasite-induced simultaneous release of other immunomodulators, which might act synergistically with IL-10 in the complex interplay to down-regulate the TNF--mediated inflammatory response. While seemingly distinct from the initially described cytokine synthesis inhibitory function of IL-10, the efficacy of this cytokine in attenuating leishmanicidal activity is both complementarily and mechanistically related.

It has been reported that IL-10 as a potential immunomodulator attenuates cell-mediated immune response, which in turn aids the intracellular survival of microorganisms (65). We observed that prior incubation with anti-IL-10 MAb did not affect the parasite uptake but markedly inhibited the parasite multiplication (Fig. 7). This result suggests that under parasitic stress the endogenous release of IL-10 by the host macrophage confers a beneficial role in the parasite's intracellular survival and multiplication.

Recently, it has been reported that PKC-, a Ca²⁺-dependent PKC isoform, regulates innate macrophage functions involved in the control of infection by intracellular L. donovani (62). Our observations collectively imply that a high level of IL-10 secretion by infected macrophages down-regulates the expression and activity of Ca²⁺-dependent protein kinases and consequent inflammatory and oxidative responses. Our study also suggests that endogenous release of mIL-10 favors the intracellular survival and multiplication of L. donovani. Such a complex, multisignal regulation by IL-10 might be an adaptive strategy by the protozoan to enable it to survive within a hostile environment. Application of an anticytokine strategy to neutralize IL-10 bioactivity in vivo may be effective in the treatment of immunocompromised and/or immunocompetent patients suffering from life-threatening visceral leishmaniasis.