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. 2003 May;71(5):2439–2446. doi: 10.1128/IAI.71.5.2439-2446.2003

Immunosuppression in Hamsters with Progressive Visceral Leishmaniasis Is Associated with an Impairment of Protein Kinase C Activity in Their Lymphocytes That Can Be Partially Reversed by Okadaic Acid or Anti-Transforming Growth Factor β Antibody

Ananda Mookerjee 1, Parimal C Sen 2, Asoke C Ghose 1,*
PMCID: PMC153259  PMID: 12704114

Abstract

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual impairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC or PBMC, respectively) to in vitro stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io). Removal of macrophage-like adherent cells from SPMC or PBMC of infected animals (I-SPMC or I-PBMC) was earlier shown to restore almost completely their lymphoproliferative responses to PMA plus Io. The present study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a key role in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca2+, phosphatidyl serine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMC as well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those for normal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight in vitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in the cytosolic or membrane fraction of untreated or PMA-treated cells, respectively. Partial, though significant, restoration of PKC activity could also be achieved in the membrane fraction of PMA-treated cells following overnight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA) or an anti-transforming growth factor β (anti-TGF-β) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-β antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed to show any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms in the SPMC or PBMC of infected animals compared to those of normal controls. The results presented in this study suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressive effect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated through the downregulation of the activation pathway involving PKC and its downstream molecules such as mitogen-activated protein kinases. Further, the observed suppression of PKC activity and subsequent lymphoproliferative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylation events. The relevance of these results is discussed in relation to the role of TGF-β, levels of which are known to be elevated in visceral leishmaniasis.

Visceral leishmaniasis (VL), or kala-azar, in humans is caused by the protozoan parasite Leishmania donovani (6, 50). The disease is usually fatal if left untreated and is marked by a profound suppression of the cell-mediated immune function of the host, though plenty of circulating antibodies are demonstrable in the host's serum (9, 10, 20, 26, 27, 51). Recovery from the disease following chemotherapy is accompanied by the restoration of antigen-specific T-cell responses in vitro and in vivo, with concomitant decreases in the antibody levels (10, 26, 51). Intracardial inoculation of golden hamsters (Mesocricetus auratus) with L. donovani amastigotes also produces a progressive and fatal type of visceral disease accompanied by an impairment of the T-lymphocyte proliferative response in vitro to leishmanial antigen and mitogens such as concanavalin A (ConA) (14, 22, 35, 41). Although this defect is partly attributed to the generation of adherent cells or “suppressor macrophages” in the infected host (42), the mechanism by which such suppression is mediated remains largely unknown. Production of nitric oxide or prostaglandin E2 by these adherent cells has been shown to contribute only marginally to the observed impairment of the lymphoproliferative process (15).

Recent studies with the murine model of leishmaniasis have attributed the altered T-cell-mediated immune status of the host to defects in the transmembrane signaling mechanism, which plays a crucial role in T-cell activation (2). Thus, modulation of costimulatory signals, provided through the interaction of the CD28 and B7 molecules, appears to downregulate the antileishmanial T-cell response (38). This is believed to be mediated through the negative costimulatory receptor CTLA4, the cross-linking of which leads to increased synthesis of transforming growth factor β (TGF-β), a potent inhibitory cytokine (24). Earlier studies on hamster VL have demonstrated impairment of the proliferative response of splenic and peripheral blood mononuclear cells (SPMC and PBMC, respectively) to in vitro stimulation with a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) (15). Removal of macrophage-like adherent cells from SPMC and PBMC of infected animals, however, significantly restored their proliferative responses to PMA plus Io. While PMA is known to induce the translocation of conventional protein kinase C (c-PKC) isotypes from the cytosol to the plasma membrane and their subsequent activation, Io (a calcium ionophore) mobilizes calcium into the cell (28, 32, 57). Since isotypes of c-PKC are known to play a critical role in the T-cell activation process (1, 16, 43), it was of considerable interest to determine their status in the lymphocyte population derived from L. donovani-infected animals.

In the present study, we have investigated the activation and translocation events of PKC in the nonadherent cell populations of SPMC and PBMC from infected animals following their in vitro treatment with PMA (before and after removal of the adherent cell population). We have also determined the levels of c-PKC isotypes in these cells from infected hamsters and compared the results with those for normal (uninfected) animals. Furthermore, we have studied the effects of okadaic acid (OA), an inhibitor of Ser/Thr phosphatases (13), and anti-TGF-β on the restoration of PKC activity as well as the proliferative responses of lymphocytes from infected hamsters.

MATERIALS AND METHODS

L. donovani infection in hamsters.

Laboratory-bred strains of golden (Syrian) hamsters (M. auratus) were obtained from the National Centre for Animal Sciences, Hyderabad, India. Hamsters (6 to 8 weeks old) were intracardially inoculated with ∼2 × 107 amastigotes of L. donovani strain BI2302 (19) per 0.2 ml per animal. The infection produced progressive illness resulting in death of the animals, usually 8 to 10 weeks after inoculation. Degrees of parasitemia in the spleens and livers of infected animals were determined by sacrificing the animals at appropriate time intervals following infection and counting the parasites in the organ impression smears (54).

Preparation of mononuclear cells.

Hamsters (normal or infected) were euthanized, and their spleens were removed and homogenized separately in ice-cold RPMI-1640 medium (Sigma, St. Louis, Mo.). Lymphocyte-enriched mononuclear cells were isolated by Histopaque 1077 (Sigma) density gradient centrifugation of the spleen cell suspension, washed, and finally resuspended in cold RPMI-1640 medium supplemented with 15% heat-inactivated fetal bovine serum (RPMI-FBS). Cell viability (>95%) was checked by the trypan blue dye exclusion method. The percentage of lymphocytes present in the SPMC was found to be >95% for normal animals, while it usually varied between 70 and 90% for animals with VL depending on the progression of infection. For certain experiments, the SPMC suspension, thus obtained, was kept in 35-mm-diameter plastic tissue culture plates for about 4 h at 37°C under a 5% CO2-95% air atmosphere to allow attachment of adherent cells. Nonadherent cells (≥95% lymphocytes) were subsequently removed by aspiration, harvested by centrifugation, and resuspended in RPMI-FBS. Mononuclear cells were also isolated from heparinized blood of sacrificed animals by Histopaque gradient centrifugation. Whenever needed, adherent cells present in the PBMC preparation were removed by the plate adherence method as described earlier. Percentages of nonadherent cells (lymphocytes) in the PBMC preparation from infected hamsters before and after removal of the adherent cell population were 75 to 85% and >95%, respectively.

Lymphocyte transformation experiments.

Lymphocyte transformation experiments were carried out in vitro in 96-well tissue culture plates, each well of which contained 2 × 105 cells in 200 μl of culture. Cells were stimulated with ConA (2.5 μg/ml) or with a combination of PMA (20 ng/ml) and Io (500 ng/ml) for 48 h at 37°C under 5% CO2-95% air. Unstimulated (control) cultures did not receive any ConA or PMA plus Io. Next, cell suspensions were pulsed with [3H]thymidine (0.5 μCi/well) (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) for another 20 h. Cells were harvested on glass fiber filter papers (Whatman, Maidstone, United Kingdom) by using a cell harvester (Nunc, Roskilde, Denmark), and incorporation of [3H]thymidine was measured by a liquid scintillation counter. In some experiments, proliferation of SPMC and PBMC was studied in the presence of 20 nM OA (Gibco-BRL, Gaithersburg, Md.) or anti-TGF-β neutralizing antibody (R&D Systems, Minneapolis, Minn.), used at a final concentration of 30 μg/ml.

Preparation of membrane (particulate) and cytosolic (soluble) fractions of SPMC and PBMC.

Mononuclear cells (SPMC or PBMC) obtained from individual animals (normal or infected) were suspended in RPMI-FBS medium, distributed into 35-mm-diameter plastic tissue culture petri dishes, and cultured for 4 h at 37°C in 5% CO2-95% air. Next, cells in one set of the petri dishes were treated with PMA at a final concentration of 100 or 50 ng/ml for SPMC or PBMC, respectively. Cells in the other set of petri dishes did not receive any PMA treatment. After 15 min of further incubation at 30°C, nonadherent cells from both PMA-treated and untreated (control) petri dishes were collected and centrifuged at 4°C. Cell pellets were immediately resuspended in chilled lysing buffer containing 20 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, 1 mM EDTA, 0.1% (vol/vol) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 μg of N-p-tosyl-l-phenylalanine chloromethyl ketone/ml, 5 μg of leupeptin/ml, and 5 μg of aprotinin/ml. The cell suspension was placed in an ice bath and lysed by sonication, and the sonicated material was centrifuged initially at 500 × g and subsequently at 10,000 × g (for 10 min each time, at 4°C) to remove the debris. The supernatant was again centrifuged at 100,000 × g for 60 min at 4°C, and the clear supernatant was collected as the cytosolic (soluble) fraction. The pellet containing the membrane (particulate) fraction was resuspended in the lysing buffer (described above). Protein contents of the soluble and particulate fractions were determined by using the Bradford reagent (4). In some experiments, SPMC and PBMC from infected animals (I-SPMC and I-PBMC) were incubated overnight either with 20 nM OA or with anti-TGF-β (30 μg/ml) and subsequently treated with PMA (100 ng/ml for SPMC and 50 ng/ml for PBMC) for 15 min prior to harvesting of nonadherent cells for preparation of their membrane fraction as described earlier.

In an alternative experimental approach, nonadherent cells were initially separated from adherent cells after 4 h of culture of I-SPMC and I-PBMC in plastic petri dishes and were subsequently cultured overnight at 37°C in 5% CO2-95% air. Then these cells were either left untreated or treated with PMA for 15 min, after which their membrane and cytosolic fractions were obtained as described earlier. In parallel experiments, I-SPMC and I-PBMC (containing both nonadherent and adherent cells) were cultured overnight, after which nonadherent cells were separated and collected. Membrane and cytosolic fractions were obtained from nonadherent cells with or without prior PMA treatment (15 min).

Measurement of PKC activity.

The PKC activity in the membrane (particulate) or cytosolic (soluble) fraction was measured by using the methodology of Kikkawa et.al (31), with minor modifications. The particulate fraction was solubilized with 0.5% (vol/vol) Triton X-100 before being used for the assay. Briefly, aliquots (in triplicate) of the particulate or soluble fraction were taken in 50 μl (final volume) of the reaction mixture consisting of the following: 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM CaCl2, 0.02 mg of phosphatidyl serine (PS; Sigma)/ml, 0.005 mg of diacyl glycerol (DAG; Sigma)/ml, 0.2 mg of histone IIIS (Sigma)/ml, and 10 mM ATP containing [γ-32P]ATP (Bhaba Atomic Research Centre, Mumbai, India) to a final concentration of 1 μCi/50 μl of assay mixture. For all measurements of PKC activity, control sets were run with aliquots of the test fraction taken in the reaction mixture without CaCl2, PS, and DAG. The phosphorylation reaction was initiated by addition of ATP and was allowed to continue for 15 min at 30°C. Thereafter, the reaction was terminated by pipetting out and spotting of 40 μl of the reaction mixture onto Whatman P81 chromatography paper (2 cm2) and subsequent transfer of the spotted paper strips to a solution of 75 mM orthophosphoric acid. Strips were then washed three times in 75 mM orthophosphoric acid under mild shaking conditions, after which radioactivity was measured by a liquid scintillation counter. Results were analyzed by subtraction of the control set counts from those of the experimental sets and were expressed as picomoles of phosphate incorporated per milligram of protein per 15 min. The PKC activity of the membrane fraction was also determined similarly following overnight incubation of cells with 20 nM OA or anti-TGF-β neutralizing antibody (30 μg/ml).

Immunoblot experiments.

c-PKC levels in nonadherent SPMC and PBMC of normal and infected hamsters were determined by immunoblot experiments using either (i) a mouse monoclonal antibody directed against PKCα, -βI, -βII, and -γ (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) or (ii) a monoclonal anti-PKCα antibody (BD Biosciences, San Diego, Calif.). Levels of a constitutively expressed protein (α-actin) was determined by immunoblot experiments using a monoclonal antibody directed against α-actin (Calbiochem, La Jolla, Calif.). Briefly, whole-cell lysates of nonadherent SPMC and PBMC (prepared by sonication as described earlier) were directly used as test materials for immunoblotting. In some experiments, SPMC and PBMC from normal or infected animals were treated with PMA (50 ng/ml for PBMC and 100 ng/ml for SPMC) for 15 min prior to separation of nonadherent cells from adherent cells. The PMA-treated nonadherent cells, thus isolated, were lysed by sonication, and membrane (particulate) and cytosolic (soluble) fractions were obtained as described earlier and subjected to electroblotting using an anti-PKCα antibody. For determination of levels of mitogen-activated protein kinases (MAPK), nonadherent PMA-treated SPMC and PBMC from normal and infected hamsters were directly placed in 2× Laemmli buffer (immediately after their separation from adherent cells), followed by mild sonication of the cell lysate. The homogenates were directly used in immunoblotting experiments, in which they were probed by a polyclonal antibody directed against p42/44 MAPK (also called the extracellular signal-regulated kinases, ERK2 and ERK1) (Biolabs, Beverly, Mass.) and by an anti-phospho-p42/44 MAPK polyclonal antibody (Biolabs).

RESULTS

PKC activities in SPMC and PBMC of normal and L. donovani-infected hamsters.

PKC activities in the membrane and cytosolic fractions of nonadherent SPMC and PBMC from normal as well as L. donovani infected hamsters were determined immediately following removal of adherent cells. Results are presented in Fig. 1. While the PKC activity in untreated cells from normal hamsters was primarily associated with the cytosolic fraction, prior in vitro PMA treatment of SPMC and PBMC (containing both adherent and nonadherent cells) resulted in transfer of such activity from the cytosol to the membrane fraction of the cells (nonadherent). On the other hand, significant reductions in PKC activity were noted in the cytosolic fraction of untreated cells obtained from 30-day- or 45-day-infected animals (P < 0.001 for both groups). Significant decreases in PKC activity were also evident in the membrane fraction of PMA-treated cells from 30-day- or 45-day-infected animals (P < 0.001 for both groups) relative to that for the normal control.

FIG. 1.

FIG. 1.

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PKC activities in membrane and cytosolic fractions of untreated and PMA-treated nonadherent SPMC and PBMC from normal and infected hamsters. Enzyme activity was measured in nonadherent cells immediately after their separation from adherent cells. Results are means ± standard errors from five independent sets of experiments.

Effect of removal of adherent cell populations from SPMC and PBMC of infected hamsters on the restoration of PKC activity.

The role of the adherent cell populations present in the SPMC or PBMC of infected animals in the inhibition of PKC activity was studied in vitro. As shown in Fig. 1, cytosolic or membrane-associated PKC activity in the SPMC and PBMC of infected animals was found to be present only at low levels in nonadherent cells when they were tested immediately after removal of adherent cells. However, overnight culture of these nonadherent cells, following their separation from adherent cells, led to significant levels (P < 0.001) of restoration of PKC activity in their cytosolic (soluble) fraction (Fig. 2A). Further, treatment of these overnight cultured nonadherent cells with PMA for 15 min led to translocation of this activity from the cytosolic to the membrane fraction. It may be noted, however, that overnight culture of nonadherent cells in the presence of adherent cells and measurement of the PKC activity of the former immediately after their removal from adherent cells failed to show any such restoration of PKC activity in either untreated or PMA-treated nonadherent cells (Fig. 2B).

FIG. 2.

FIG. 2.

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PKC activity in the membrane and cytosolic fractions of nonadherent SPMC and PBMC from 45-day-infected hamsters. Enzyme activity was measured in nonadherent cells cultured overnight following their separation from adherent cells (A) or immediately after their separation from adherent cells present in overnight cultures of SPMC and PBMC (B). Results are means ± standard errors from four independent sets of experiments.

Demonstration of PKC in SPMC and PBMC by immunoblot analysis.

c-PKC levels in nonadherent SPMC and PBMC of 45-day-infected hamsters were determined by immunoblot experiments (Fig. 3), and the data were compared with those obtained for normal (uninfected) animals. Figure 3A shows immunoblot results obtained with the antibody directed against c-PKC isoforms (α, βI, βII, and γ). Similar data were generated by using an antiserum specific for PKCα (Fig. 3B), which was found to be the predominant c-PKC isoform present in the lymphocytes of hamsters (data not shown). Also included in Fig. 3 is the immunoblot data generated with the same preparations by an antibody recognizing the constitutively expressed (control) protein α-actin (Fig. 3C). Densitometric analyses of the data, plotted as ratios of the PKC isoform(s) versus control protein bands, are shown on the right. The data suggest that there is no decrease in the levels of these PKC isoforms in nonadherent SPMC and PBMC of 45-day-infected hamsters relative to those for uninfected (normal) animals. The demonstration that the levels of c-PKC in nonadherent SPMC and PBMC of infected animals remained essentially undiminished prompted us to determine their status following PMA treatment of cells. Immunoblot results show that PMA treatment of SPMC and PBMC led to translocation of c-PKC from the cytosol to the membrane in nonadherent cells from both normal and infected hamsters. This was true for the data obtained with the α-isoform-specific antiserum (Fig. 4) as well as for results obtained with the monoclonal antibody specific for the α, βI, βII, and γ isoforms (data not shown) that was used to probe c-PKC isoforms in immunoblot experiments.

FIG. 3.

FIG. 3.

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(Left) Immunoblot analyses of c-PKC (A) and PKCα (B) in nonadherent SPMC and PBMC of normal (N) and 45-day-infected (I) hamsters. Nonadherent cells were processed immediately after their separation from adherent cells. Each lane was loaded with 50 μg of whole-cell lysate protein, and the blot was developed using either a monoclonal antibody to different isotypes of c-PKC (A) or a monoclonal antibody to PKCα (B). Results obtained with a monoclonal antibody to the constitutively expressed protein α-actin are also shown (C) for comparison. (Right) Bar graphs present densitometric analysis results as ratios of intensities of PKC isoforms to those of α-actin bands. Results shown are representative of three independent sets of experiments involving six animals.

FIG. 4.

FIG. 4.

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Immunoblot analyses of membrane and cytosolic fractions of nonadherent SPMC and PBMC of normal (N) and 45-day-infected (I) hamsters. Membrane and cytosolic fractions were isolated from nonadherent cells of PMA-treated or untreated SPMC and PBMC. Each lane was loaded with 30 μg of protein, and the blot was developed using a monoclonal antibody to PKCα. Results are representative of three independent sets of experiments involving six animals.

The p42/44 MAPK and their phosphorylation following PMA stimulation of SPMC and PBMC from normal and infected hamsters.

p42/44 MAPK levels in nonadherent PBMC from normal and L. donovani-infected animals appear comparable (Fig. 5A). MAPK levels in nonadherent SPMC from infected hamsters, however, seem to be somewhat lower than those in nonadherent SPMC from normal animals. Interestingly, the phosphorylation status of p42/44 MAPK was found to be compromised in PMA-treated nonadherent SPMC and PBMC from infected hamsters compared to those in their normal counterparts (Fig. 5B).

FIG. 5.

FIG. 5.

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(Bottom) Immunoblot analyses of p42/44 MAPK and their activated forms in nonadherent SPMC and PBMC of normal and 45-day-infected hamsters. Each lane was loaded with lysates of 106 cells, and the blot was developed with polyclonal antibodies to p42/44 MAPK (A) or their dually phosphorylated forms (B). (Top) Bar graphs present densitometric analyses of the MAPK bands. Results are representative of two independent sets of experiments involving four animals.

Effect of OA or anti-TGF-β treatment of SPMC and PBMC on restoration of membrane-bound PKC activity.

The effect of treatment of I-SPMC and I-PBMC with OA or with an anti-TGF-β neutralizing antibody on the restoration of PKC activity was studied. The results presented in Fig. 6 demonstrate that membrane-bound PKC activity could be restored partially, though significantly (P < 0.001), in nonadherent cells following overnight culture of I-SPMC and I-PBMC in the presence of OA and subsequent PMA treatment. Restoration of PKC activity was found to be ∼59% for SPMC and ∼51% for PBMC relative to control PKC activity levels in PMA-treated cells from normal animals. Similarly, overnight treatment of I-SPMC and I-PBMC with an anti-TGF-β antibody followed by PMA treatment significantly (P < 0.001) restored membrane-bound PKC activity in nonadherent cells, to ∼45% (for SPMC) and ∼41% (for PBMC) of activity levels for normal animals.

FIG. 6.

FIG. 6.

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Effects of in vitro OA or anti-TGF-β treatment of SPMC and PBMC from 45-day-infected hamsters on restoration of PKC activity in the membrane fractions of their nonadherent cell populations. PKC activities in the membrane fractions of nonadherent SPMC and PBMC from normal hamsters are also presented for the sake of comparison. Results are means ± standard errors from three independent sets of experiments.

Effects of OA and anti-TGF-β treatment of SPMC and PBMC on restoration of lymphoproliferative response.

The effect of in vitro treatment of I-SPMC and I-PBMC with OA or anti-TGF-β on the restoration of the lymphoproliferative response was studied. The results presented in Fig. 7 demonstrate that the presence of OA in the culture medium partially, though significantly (P < 0.001), restored the proliferative responses of I-SPMC and I-PBMC to in vitro stimulation with a combination of PMA and Io. The magnitude of restoration was ∼53% for I-SPMC and ∼58% for I-PBMC relative to the proliferative responses of cells from normal animals treated with PMA plus Io only. Similarly, the presence of an anti-TGF-β neutralizing antibody in the culture medium significantly (P < 0.001) restored the proliferative responses of I-SPMC and I-PBMC to PMA-plus-Io stimulation. Thus, the magnitudes of restoration were ∼47 and ∼75% for I-SPMC and I-PBMC, respectively, relative to the response levels obtained with PMA-plus-Io-treated cells of normal animals. It may be noted that OA or anti-TGF-β treatment did not result in any significant (P > 0.05) alteration in the proliferative response of PMA-plus-Io-stimulated cultures of SPMC or PBMC from normal animals.

FIG. 7.

FIG. 7.

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Effect of in vitro OA or anti-TGF-β treatment of SPMC and PBMC from 45-day-infected hamsters on the restoration of their lymphoproliferative responses following stimulation with a combination of PMA and Io. Proliferative responses of SPMC and PBMC from normal hamsters following PMA-plus-Io stimulation are also presented for the sake of comparison. Results are means ± standard errors from three independent sets of experiments.

DISCUSSION

VL in humans is accompanied by profound suppression of T-cell responsiveness to leishmanial antigen both in vivo and in vitro (9, 10, 26, 27, 50, 51). While the exact mechanism of immunosuppression in human VL has yet to be understood clearly, studies with the murine VL model demonstrated alterations or defects of the infected host in antigen-presenting ability and/or generation of appropriate costimulatory signals which are required for T-cell activation (3, 29, 53). However, such mechanisms involving defects in the transmembrane signaling process may not be adequate to explain the generalized immunosuppression (with regard to mitogens and unrelated antigens) that has been reported from certain geographical regions (12, 26, 27, 30). Immunosuppression in hamsters with progressive VL and lack of lymphoproliferative response to in vitro stimulation with PMA plus Io also indicate an additional defect(s) in the intracellular signaling mechanism in T cells of animals, particularly at the late stage of infection (14, 15). While PMA primarily acts via a PKC-dependent pathway by activating several isoforms of the protein kinase, which include the Ca2+- and PS-dependent PKC isoforms (16), Io, a calcium ionophore, helps in the mobilization of calcium inside the cell (55). The results presented in this study demonstrate an impairment of PKC activity (Ca2+, PS, and DAG dependent) in lymphocytes derived from hamsters with progressive VL despite the fact that synthesis of the major isoform(s) of PKC remained undiminished. As a matter of fact, a marginal increase in the expression of c-PKC was noted in infected animals; its significance remains to be established.

Our results also show that translocation of c-PKC isoforms from the cytosol to the membrane (following PMA treatment) remained essentially unaffected. However, the lack of activation of PKC is also reflected in the blockade of downstream signaling pathways involved in the activation of p42/44 MAPK (52) following PMA treatment. Although the activation of p42/44 MAPK by PMA is primarily mediated through a PKC-dependent pathway, recent studies have documented evidence in favor of a PKC-independent pathway as well (47). Therefore, it is possible that both PKC-dependent and -independent signaling pathways are affected in lymphocytes derived from terminally ill hamsters with VL. Impairment of PKC (7, 37, 45, 48) as well as p42/44 MAPK (39) activity in L. donovani-infected macrophages in vitro has already been documented by several workers, although no such report is available so far on lymphocytes of infected animals. Thus, attenuation of PMA-induced activation as well as translocation of PKC was demonstrated in L. donovani-infected monocytes from human PBMC (45). However, in another study, impairment of PKC activity was documented in murine bone marrow-derived macrophages following leishmanial infection in vitro, despite the fact that the distribution and translocation of PKC isotypes remained largely unaffected (46). Although the exact mechanism underlying the observed attenuation of PKC activity or PKC-dependent gene expression was not clearly established, several structural components of leishmanial parasites (e.g., lipophosphoglycan and glycoinositol phospholipid (GIPL) were shown to downregulate the PKC activity of the host macrophages (17, 23, 34). Since these inhibitors act either by direct interaction (17, 34) with the host PKC inside the infected macrophages or indirectly by binding to their plasma membranes (only after pretreatment of cells with high concentrations of purified lipophosphoglycan) (23), they are less likely to play a major role in the observed downregulation of PKC activity in lymphocytes which do not harbor leishmanial parasites.

Several studies indicate that phosphorylation of α and β isoforms of PKC in their activation loop is essential for their priming towards activation (16, 25). Further, membrane association of PKC alone is now believed to be an insufficient criterion for determination of its activity (56). In fact, these studies have emphasized the importance of dephosphorylation by phosphatases that act on PKC and thus modulate the dynamics of the phosphorylation-dephosphorylation process in cell physiology and function (40). The results presented in this study appear to be consistent with this concept, as significant restoration of PKC activity in lymphocytes is demonstrable following pretreatment of SPMC and PBMC from infected animals with the Ser/Thr phosphatase inhibitor OA. Further, the presence of OA in the culture medium helped to achieve considerable restoration of lymphoproliferative responses of SPMC and PBMC following stimulation with PMA plus Io. We have also seen marked suppression of lymphoproliferative responses of SPMC and PBMC from infected hamsters to in vitro stimulation with ConA, which could be partially, though significantly, restored following treatment with sodium orthovanadate, an inhibitor of protein tyrosine phosphatases (PTPases) (A. Mookerjee and A. C. Ghose, unpublished data). All these results suggest the importance of phosphorylation-dephosphorylation events that, in turn, play a crucial role in the signaling process of lymphocytes leading to their activation and proliferation. Leishmanial infection probably interferes with this process by modulating these events through increased intracellular activity of phosphatases. As a matter of fact, several studies have demonstrated that Leishmania-infected murine macrophages exhibit elevated levels of PTPase activity (18, 33, 44), which may be responsible for downregulation of protein kinases and subsequent signal transduction processes. Interestingly, we have also noted higher levels of PTPase activity in lymphocytes derived from L. donovani-infected hamsters than in their normal counterparts (Mookerjee and Ghose, unpublished).

Progressive visceral infection with L. donovani in hamsters has been shown to induce synthesis of the deactivating cytokines such as interleukin-10 and TGF-β (35, 36). Further, the proliferative response of lymph node cells from hamsters immunized with leishmanial antigen could be inhibited by adherent cells obtained from spleens of L. donovani-infected hamsters, and such an inhibitory effect was abolished by an anti-TGF-β antibody (49). Increased synthesis of TGF-β in hamster VL has also been observed (P. Mukherjee, A. Mookerjee, and A. C. Ghose, unpublished data). Furthermore, we also observed downregulation of the lymphoproliferative response as well as the PKC activity of normal lymphocytes by supernatants of overnight cultures of adherent cells from infected animals (data not shown). Significant, though partial, restoration of the lymphoproliferative response as well as of PKC activity by an anti-TGF-β neutralizing antibody testifies to the importance of this deactivating cytokine for the impairment of PKC activity and the manifestation of immunosuppression in hamster VL. The importance of TGF-β in the suppression of cellular immunity and the subsequent increase in susceptibility to infection has recently been established in murine VL (58).

Several reports have demonstrated that TGF-β can act as a suppressor of cellular proliferation (5, 8, 21), which may be attributed to its ability to increase protein phosphatase activities of target cells (8, 11, 21). Further, inhibition of cell cycle progression by the TGF-β signaling pathway has been shown to be mediated through downregulation of cyclin A promoter activity, which can be reversed by OA (59). Our results suggest that a similar mechanism may be involved in hamster VL, as impairment of the lymphoproliferative response in vitro may be related to increased protein phosphatase activities, which would downregulate intracellular protein kinase activities. Thus, removal of adherent cells (the principal source of TGF-β) from the SPMC and PBMC helps in the partial restoration of both PKC activity and proliferation of lymphocytes from infected animals. Our recent data also indicate the importance of TGF-β for impairment of PKC activity as well as for impairment of the proliferative response of lymph node cells derived from hamsters with high levels of L. donovani infection (Mookerjee and Ghose, unpublished). Further studies are under way to ascertain the status of other protein kinases and phosphatases in the immunosuppression associated with VL.

Acknowledgments

This work was partly supported by a grant (SP/SO/B32/98) from the Department of Science and Technology, Government of India. A. Mookerjee is the recipient of a fellowship from the Council of Scientific and Industrial Research, Government of India.

We thank Ranjit Roy and G. Sa for their interest in this study. We acknowledge the helpful technical assistance of Prabal Gupta and Debashish Mazumdar.

Editor: W. A. Petri, Jr.

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