Immunosuppression in Hamsters with Progressive Visceral … · Visceral leishmaniasis (VL), or...

INFECTION AND IMMUNITY, May 2003, p. 2439–2446 Vol. 71, No. 50019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.5.2439–2446.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Immunosuppression in Hamsters with Progressive VisceralLeishmaniasis Is Associated with an Impairment of Protein Kinase C

Activity in Their Lymphocytes That Can Be Partially Reversed byOkadaic Acid or Anti-Transforming Growth Factor � Antibody

Ananda Mookerjee,1 Parimal C. Sen,2 and Asoke C. Ghose1*Departments of Microbiology1 and Chemistry,2 Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India

Received 11 October 2002/Returned for modification 25 November 2002/Accepted 30 January 2003

Progressive visceral infection of golden hamsters by Leishmania donovani amastigotes led to gradual im-pairment of the proliferative responses of their splenic or peripheral blood mononuclear cells (SPMC orPBMC, 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. Thepresent study was directed to evaluate the status of protein kinase C (PKC), a molecule(s) known to play a keyrole in the lymphoproliferative process. Our results demonstrate that PKC activities (Ca2�, phosphatidylserine, and diacyl glycerol dependent) in the cytosolic fraction of untreated nonadherent I-SPMC or I-PBMCas well as in the membrane fraction of PMA-treated cells were decreased significantly relative to those fornormal controls. However, removal of adherent cells from I-SPMC or I-PBMC and subsequent overnight invitro cultivation of nonadherent cells (lymphocytes) resulted in significant restoration of PKC activity in thecytosolic 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 followingovernight in vitro treatment of I-SPMC or I-PBMC with the Ser/Thr phosphatase inhibitor okadaic acid (OA)or an anti-transforming growth factor � (anti-TGF-�) neutralizing antibody. These results correlated well withthe ability of OA or the anti-TGF-� antibody to restore the lymphoproliferative response of I-SPMC or I-PBMC following stimulation with PMA plus Io. Interestingly enough, immunoblotting experiments failed toshow any reduction in the level or translocation (following PMA treatment) of conventional PKC isoforms inthe SPMC or PBMC of infected animals compared to those of normal controls. The results presented in thisstudy suggest that the adherent cells generated in the SPMC or PBMC of infected animals exert a suppressiveeffect on the proliferative response of nonadherent cells (lymphocytes) which is likely to be mediated throughthe 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 lymphoprolif-erative responses can be attributed to alternations in the intracellular phosphorylation-dephosphorylationevents. The relevance of these results is discussed in relation to the role of TGF-�, levels of which are knownto be elevated in visceral leishmaniasis.

Visceral leishmaniasis (VL), or kala-azar, in humans iscaused by the protozoan parasite Leishmania donovani (6, 50).The disease is usually fatal if left untreated and is marked by aprofound suppression of the cell-mediated immune function ofthe host, though plenty of circulating antibodies are demon-strable in the host’s serum (9, 10, 20, 26, 27, 51). Recovery fromthe disease following chemotherapy is accompanied by therestoration of antigen-specific T-cell responses in vitro and invivo, with concomitant decreases in the antibody levels (10, 26,51). Intracardial inoculation of golden hamsters (Mesocricetusauratus) with L. donovani amastigotes also produces a progres-sive and fatal type of visceral disease accompanied by an im-pairment of the T-lymphocyte proliferative response in vitro toleishmanial antigen and mitogens such as concanavalin A(ConA) (14, 22, 35, 41). Although this defect is partly attrib-uted to the generation of adherent cells or “suppressor mac-

rophages” in the infected host (42), the mechanism by whichsuch suppression is mediated remains largely unknown. Pro-duction of nitric oxide or prostaglandin E2 by these adherentcells has been shown to contribute only marginally to the ob-served impairment of the lymphoproliferative process (15).

Recent studies with the murine model of leishmaniasis haveattributed the altered T-cell-mediated immune status of thehost to defects in the transmembrane signaling mechanism,which plays a crucial role in T-cell activation (2). Thus, mod-ulation of costimulatory signals, provided through the interac-tion of the CD28 and B7 molecules, appears to downregulatethe antileishmanial T-cell response (38). This is believed to bemediated through the negative costimulatory receptor CTLA4,the cross-linking of which leads to increased synthesis of trans-forming growth factor � (TGF-�), a potent inhibitory cytokine(24). Earlier studies on hamster VL have demonstrated im-pairment of the proliferative response of splenic and periph-eral blood mononuclear cells (SPMC and PBMC, respectively)to in vitro stimulation with a combination of phorbol 12-my-ristate 13-acetate (PMA) and ionomycin (Io) (15). Removal ofmacrophage-like adherent cells from SPMC and PBMC of

* Corresponding author. Mailing address: Department of Microbi-ology, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054,India. Phone: 91 33 2337-9416, -9544, or -9219. Fax: 91 33 2334-3886.E-mail: [email protected].

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infected animals, however, significantly restored their prolifer-ative responses to PMA plus Io. While PMA is known toinduce the translocation of conventional protein kinase C (c-PKC) isotypes from the cytosol to the plasma membrane andtheir subsequent activation, Io (a calcium ionophore) mobi-lizes calcium into the cell (28, 32, 57). Since isotypes of c-PKCare known to play a critical role in the T-cell activation process(1, 16, 43), it was of considerable interest to determine theirstatus in the lymphocyte population derived from L. donovani-infected animals.

In the present study, we have investigated the activation andtranslocation events of PKC in the nonadherent cell popula-tions of SPMC and PBMC from infected animals followingtheir in vitro treatment with PMA (before and after removal ofthe adherent cell population). We have also determined thelevels of c-PKC isotypes in these cells from infected hamstersand compared the results with those for normal (uninfected)animals. Furthermore, we have studied the effects of okadaicacid (OA), an inhibitor of Ser/Thr phosphatases (13), andanti-TGF-� on the restoration of PKC activity as well as theproliferative 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 AnimalSciences, Hyderabad, India. Hamsters (6 to 8 weeks old) were intracardiallyinoculated with �2 � 107 amastigotes of L. donovani strain BI2302 (19) per 0.2ml per animal. The infection produced progressive illness resulting in death ofthe animals, usually 8 to 10 weeks after inoculation. Degrees of parasitemia inthe spleens and livers of infected animals were determined by sacrificing theanimals at appropriate time intervals following infection and counting the par-asites in the organ impression smears (54).

Preparation of mononuclear cells. Hamsters (normal or infected) were eutha-nized, and their spleens were removed and homogenized separately in ice-coldRPMI-1640 medium (Sigma, St. Louis, Mo.). Lymphocyte-enriched mononu-clear cells were isolated by Histopaque 1077 (Sigma) density gradient centrifu-gation of the spleen cell suspension, washed, and finally resuspended in coldRPMI-1640 medium supplemented with 15% heat-inactivated fetal bovine se-rum (RPMI-FBS). Cell viability (�95%) was checked by the trypan blue dyeexclusion method. The percentage of lymphocytes present in the SPMC wasfound to be �95% for normal animals, while it usually varied between 70 and90% for animals with VL depending on the progression of infection. For certainexperiments, the SPMC suspension, thus obtained, was kept in 35-mm-diameterplastic tissue culture plates for about 4 h at 37°C under a 5% CO2–95% airatmosphere to allow attachment of adherent cells. Nonadherent cells (�95%lymphocytes) were subsequently removed by aspiration, harvested by centrifu-gation, and resuspended in RPMI-FBS. Mononuclear cells were also isolatedfrom heparinized blood of sacrificed animals by Histopaque gradient centrifu-gation. Whenever needed, adherent cells present in the PBMC preparation wereremoved by the plate adherence method as described earlier. Percentages ofnonadherent cells (lymphocytes) in the PBMC preparation from infected ham-sters before and after removal of the adherent cell population were 75 to 85%and �95%, respectively.

Lymphocyte transformation experiments. Lymphocyte transformation exper-iments were carried out in vitro in 96-well tissue culture plates, each well of whichcontained 2 � 105 cells in 200 �l of culture. Cells were stimulated with ConA (2.5�g/ml) or with a combination of PMA (20 ng/ml) and Io (500 ng/ml) for 48 h at37°C under 5% CO2–95% air. Unstimulated (control) cultures did not receiveany ConA or PMA plus Io. Next, cell suspensions were pulsed with [3H]thymi-dine (0.5 �Ci/well) (Amersham, Little Chalfont, Buckinghamshire, UnitedKingdom) for another 20 h. Cells were harvested on glass fiber filter papers(Whatman, Maidstone, United Kingdom) by using a cell harvester (Nunc, Rosk-ilde, Denmark), and incorporation of [3H]thymidine was measured by a liquidscintillation counter. In some experiments, proliferation of SPMC and PBMCwas studied in the presence of 20 nM OA (Gibco-BRL, Gaithersburg, Md.) oranti-TGF-� neutralizing antibody (R&D Systems, Minneapolis, Minn.), used ata final concentration of 30 �g/ml.

Preparation of membrane (particulate) and cytosolic (soluble) fractions ofSPMC and PBMC. Mononuclear cells (SPMC or PBMC) obtained from indi-vidual animals (normal or infected) were suspended in RPMI-FBS medium,distributed into 35-mm-diameter plastic tissue culture petri dishes, and culturedfor 4 h at 37°C in 5% CO2–95% air. Next, cells in one set of the petri dishes weretreated with PMA at a final concentration of 100 or 50 ng/ml for SPMC orPBMC, respectively. Cells in the other set of petri dishes did not receive anyPMA treatment. After 15 min of further incubation at 30°C, nonadherent cellsfrom both PMA-treated and untreated (control) petri dishes were collected andcentrifuged at 4°C. Cell pellets were immediately resuspended in chilled lysingbuffer 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 �g of N-p-tosyl-L-phenylalanine chloromethyl ketone/ml, 5 �g of leupeptin/ml, and 5 �g ofaprotinin/ml. The cell suspension was placed in an ice bath and lysed by sonica-tion, and the sonicated material was centrifuged initially at 500 � g and subse-quently at 10,000 � g (for 10 min each time, at 4°C) to remove the debris. Thesupernatant was again centrifuged at 100,000 � g for 60 min at 4°C, and the clearsupernatant was collected as the cytosolic (soluble) fraction. The pellet contain-ing the membrane (particulate) fraction was resuspended in the lysing buffer(described above). Protein contents of the soluble and particulate fractions weredetermined by using the Bradford reagent (4). In some experiments, SPMC andPBMC from infected animals (I-SPMC and I-PBMC) were incubated overnighteither with 20 nM OA or with anti-TGF-� (30 �g/ml) and subsequently treatedwith PMA (100 ng/ml for SPMC and 50 ng/ml for PBMC) for 15 min prior toharvesting of nonadherent cells for preparation of their membrane fraction asdescribed earlier.

In an alternative experimental approach, nonadherent cells were initially sep-arated from adherent cells after 4 h of culture of I-SPMC and I-PBMC in plasticpetri 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 describedearlier. In parallel experiments, I-SPMC and I-PBMC (containing both nonad-herent and adherent cells) were cultured overnight, after which nonadherentcells were separated and collected. Membrane and cytosolic fractions were ob-tained from nonadherent cells with or without prior PMA treatment (15 min).

Measurement of PKC activity. The PKC activity in the membrane (particu-late) or cytosolic (soluble) fraction was measured by using the methodology ofKikkawa et.al (31), with minor modifications. The particulate fraction was solu-bilized 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 �l(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 [�-32P]ATP (Bhaba Atomic ResearchCentre, Mumbai, India) to a final concentration of 1 �Ci/50 �l of assay mixture.For all measurements of PKC activity, control sets were run with aliquots of thetest fraction taken in the reaction mixture without CaCl2, PS, and DAG. Thephosphorylation reaction was initiated by addition of ATP and was allowed tocontinue for 15 min at 30°C. Thereafter, the reaction was terminated by pipettingout and spotting of 40 �l of the reaction mixture onto Whatman P81 chroma-tography paper (2 cm2) and subsequent transfer of the spotted paper strips to asolution of 75 mM orthophosphoric acid. Strips were then washed three times in75 mM orthophosphoric acid under mild shaking conditions, after which radio-activity was measured by a liquid scintillation counter. Results were analyzed bysubtraction of the control set counts from those of the experimental sets andwere expressed as picomoles of phosphate incorporated per milligram of proteinper 15 min. The PKC activity of the membrane fraction was also determinedsimilarly following overnight incubation of cells with 20 nM OA or anti-TGF-�neutralizing antibody (30 �g/ml).

Immunoblot experiments. c-PKC levels in nonadherent SPMC and PBMC ofnormal and infected hamsters were determined by immunoblot experimentsusing either (i) a mouse monoclonal antibody directed against PKC�, -�I, -�II,and -� (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) or (ii) a monoclonalanti-PKC� antibody (BD Biosciences, San Diego, Calif.). Levels of a constitu-tively expressed protein (�-actin) was determined by immunoblot experimentsusing a monoclonal antibody directed against �-actin (Calbiochem, La Jolla,Calif.). Briefly, whole-cell lysates of nonadherent SPMC and PBMC (preparedby sonication as described earlier) were directly used as test materials for im-munoblotting. In some experiments, SPMC and PBMC from normal or infectedanimals 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. ThePMA-treated nonadherent cells, thus isolated, were lysed by sonication, andmembrane (particulate) and cytosolic (soluble) fractions were obtained as de-

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scribed earlier and subjected to electroblotting using an anti-PKC� antibody. Fordetermination of levels of mitogen-activated protein kinases (MAPK), nonad-herent PMA-treated SPMC and PBMC from normal and infected hamsters weredirectly placed in 2� Laemmli buffer (immediately after their separation fromadherent cells), followed by mild sonication of the cell lysate. The homogenateswere directly used in immunoblotting experiments, in which they were probed bya polyclonal antibody directed against p42/44 MAPK (also called the extracel-lular signal-regulated kinases, ERK2 and ERK1) (Biolabs, Beverly, Mass.) andby an anti-phospho-p42/44 MAPK polyclonal antibody (Biolabs).

RESULTS

PKC activities in SPMC and PBMC of normal and L. do-novani-infected hamsters. PKC activities in the membrane andcytosolic fractions of nonadherent SPMC and PBMC fromnormal as well as L. donovani infected hamsters were deter-mined immediately following removal of adherent cells. Re-sults are presented in Fig. 1. While the PKC activity in un-treated cells from normal hamsters was primarily associatedwith the cytosolic fraction, prior in vitro PMA treatment ofSPMC and PBMC (containing both adherent and nonadherentcells) resulted in transfer of such activity from the cytosol tothe membrane fraction of the cells (nonadherent). On theother hand, significant reductions in PKC activity were notedin 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 themembrane fraction of PMA-treated cells from 30-day- or 45-day-infected animals (P 0.001 for both groups) relative tothat for the normal control.

Effect of removal of adherent cell populations from SPMCand PBMC of infected hamsters on the restoration of PKCactivity. The role of the adherent cell populations present inthe SPMC or PBMC of infected animals in the inhibition ofPKC activity was studied in vitro. As shown in Fig. 1, cytosolicor membrane-associated PKC activity in the SPMC and PBMCof infected animals was found to be present only at low levelsin nonadherent cells when they were tested immediately afterremoval of adherent cells. However, overnight culture of thesenonadherent cells, following their separation from adherentcells, led to significant levels (P 0.001) of restoration of PKCactivity in their cytosolic (soluble) fraction (Fig. 2A). Further,treatment of these overnight cultured nonadherent cells withPMA for 15 min led to translocation of this activity from thecytosolic to the membrane fraction. It may be noted, however,that overnight culture of nonadherent cells in the presence ofadherent cells and measurement of the PKC activity of theformer immediately after their removal from adherent cellsfailed to show any such restoration of PKC activity in eitheruntreated or PMA-treated nonadherent cells (Fig. 2B).

Demonstration of PKC in SPMC and PBMC by immunoblotanalysis. c-PKC levels in nonadherent SPMC and PBMC of45-day-infected hamsters were determined by immunoblotexperiments (Fig. 3), and the data were compared with thoseobtained for normal (uninfected) animals. Figure 3A showsimmunoblot results obtained with the antibody directedagainst c-PKC isoforms (�, �I, �II, and �). Similar data weregenerated by using an antiserum specific for PKC� (Fig. 3B),which was found to be the predominant c-PKC isoform presentin the lymphocytes of hamsters (data not shown). Also in-cluded in Fig. 3 is the immunoblot data generated with thesame preparations by an antibody recognizing the constitu-tively expressed (control) protein �-actin (Fig. 3C). Densito-metric analyses of the data, plotted as ratios of the PKC iso-form(s) versus control protein bands, are shown on the right.The data suggest that there is no decrease in the levels of thesePKC 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 non-adherent SPMC and PBMC of infected animals remained es-sentially undiminished prompted us to determine their statusfollowing PMA treatment of cells. Immunoblot results showthat PMA treatment of SPMC and PBMC led to translocationof c-PKC from the cytosol to the membrane in nonadherentcells from both normal and infected hamsters. This was truefor the data obtained with the �-isoform-specific antiserum(Fig. 4) as well as for results obtained with the monoclonalantibody specific for the �, �I, �II, and � isoforms (data notshown) that was used to probe c-PKC isoforms in immunoblotexperiments.

The p42/44 MAPK and their phosphorylation followingPMA stimulation of SPMC and PBMC from normal and in-fected hamsters. p42/44 MAPK levels in nonadherent PBMCfrom normal and L. donovani-infected animals appear compa-rable (Fig. 5A). MAPK levels in nonadherent SPMC from

FIG. 1. PKC activities in membrane and cytosolic fractions of un-treated and PMA-treated nonadherent SPMC and PBMC from nor-mal and infected hamsters. Enzyme activity was measured in nonad-herent cells immediately after their separation from adherent cells.Results are means standard errors from five independent sets ofexperiments.

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infected hamsters, however, seem to be somewhat lower thanthose in nonadherent SPMC from normal animals. Interest-ingly, the phosphorylation status of p42/44 MAPK was foundto be compromised in PMA-treated nonadherent SPMC andPBMC from infected hamsters compared to those in theirnormal counterparts (Fig. 5B).

Effect of OA or anti-TGF-� treatment of SPMC and PBMCon restoration of membrane-bound PKC activity. The effect oftreatment of I-SPMC and I-PBMC with OA or with an anti-TGF-� neutralizing antibody on the restoration of PKC activ-ity was studied. The results presented in Fig. 6 demonstratethat membrane-bound PKC activity could be restored partially,though significantly (P 0.001), in nonadherent cells followingovernight culture of I-SPMC and I-PBMC in the presence ofOA and subsequent PMA treatment. Restoration of PKC ac-tivity was found to be �59% for SPMC and �51% for PBMCrelative to control PKC activity levels in PMA-treated cellsfrom normal animals. Similarly, overnight treatment of I-SPMCand I-PBMC with an anti-TGF-� antibody followed by PMAtreatment significantly (P 0.001) restored membrane-boundPKC activity in nonadherent cells, to �45% (for SPMC) and�41% (for PBMC) of activity levels for normal animals.

Effects of OA and anti-TGF-� treatment of SPMC andPBMC on restoration of lymphoproliferative response. Theeffect of in vitro treatment of I-SPMC and I-PBMC with OA oranti-TGF-� on the restoration of the lymphoproliferative re-sponse was studied. The results presented in Fig. 7 demon-strate that the presence of OA in the culture medium partially,though significantly (P 0.001), restored the proliferative re-sponses of I-SPMC and I-PBMC to in vitro stimulation with acombination of PMA and Io. The magnitude of restoration was�53% for I-SPMC and �58% for I-PBMC relative to theproliferative responses of cells from normal animals treatedwith PMA plus Io only. Similarly, the presence of an anti-TGF-� neutralizing antibody in the culture medium signifi-cantly (P 0.001) restored the proliferative responses ofI-SPMC and I-PBMC to PMA-plus-Io stimulation. Thus, themagnitudes of restoration were �47 and �75% for I-SPMCand I-PBMC, respectively, relative to the response levels ob-tained with PMA-plus-Io-treated cells of normal animals. Itmay be noted that OA or anti-TGF-� treatment did not resultin any significant (P � 0.05) alteration in the proliferativeresponse of PMA-plus-Io-stimulated cultures of SPMC orPBMC from normal animals.

FIG. 2. PKC activity in the membrane and cytosolic fractions of nonadherent SPMC and PBMC from 45-day-infected hamsters. Enzymeactivity was measured in nonadherent cells cultured overnight following their separation from adherent cells (A) or immediately after theirseparation from adherent cells present in overnight cultures of SPMC and PBMC (B). Results are means standard errors from four independentsets of experiments.



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DISCUSSION

VL in humans is accompanied by profound suppression ofT-cell responsiveness to leishmanial antigen both in vivo and invitro (9, 10, 26, 27, 50, 51). While the exact mechanism ofimmunosuppression in human VL has yet to be understoodclearly, studies with the murine VL model demonstrated alter-ations or defects of the infected host in antigen-presentingability and/or generation of appropriate costimulatory signalswhich are required for T-cell activation (3, 29, 53). However,such mechanisms involving defects in the transmembrane sig-naling process may not be adequate to explain the generalizedimmunosuppression (with regard to mitogens and unrelatedantigens) that has been reported from certain geographicalregions (12, 26, 27, 30). Immunosuppression in hamsters withprogressive VL and lack of lymphoproliferative response to invitro stimulation with PMA plus Io also indicate an additionaldefect(s) in the intracellular signaling mechanism in T cells ofanimals, particularly at the late stage of infection (14, 15).While PMA primarily acts via a PKC-dependent pathway byactivating several isoforms of the protein kinase, which includethe Ca2�- and PS-dependent PKC isoforms (16), Io, a calciumionophore, helps in the mobilization of calcium inside the cell(55). The results presented in this study demonstrate an im-pairment of PKC activity (Ca2�, PS, and DAG dependent) inlymphocytes derived from hamsters with progressive VL de-spite the fact that synthesis of the major isoform(s) of PKCremained undiminished. As a matter of fact, a marginal in-crease in the expression of c-PKC was noted in infected ani-mals; its significance remains to be established.

Our results also show that translocation of c-PKC isoformsfrom the cytosol to the membrane (following PMA treatment)remained essentially unaffected. However, the lack of activa-tion of PKC is also reflected in the blockade of downstreamsignaling pathways involved in the activation of p42/44 MAPK

(52) following PMA treatment. Although the activation ofp42/44 MAPK by PMA is primarily mediated through a PKC-dependent pathway, recent studies have documented evidencein favor of a PKC-independent pathway as well (47). There-fore, it is possible that both PKC-dependent and -independentsignaling pathways are affected in lymphocytes derived fromterminally 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 byseveral workers, although no such report is available so far onlymphocytes of infected animals. Thus, attenuation of PMA-induced activation as well as translocation of PKC was dem-onstrated in L. donovani-infected monocytes from humanPBMC (45). However, in another study, impairment of PKCactivity was documented in murine bone marrow-derived mac-rophages following leishmanial infection in vitro, despite the

FIG. 4. Immunoblot analyses of membrane and cytosolic fractionsof nonadherent SPMC and PBMC of normal (N) and 45-day-infected(I) hamsters. Membrane and cytosolic fractions were isolated fromnonadherent cells of PMA-treated or untreated SPMC and PBMC.Each lane was loaded with 30 �g of protein, and the blot was devel-oped using a monoclonal antibody to PKC�. Results are representativeof three independent sets of experiments involving six animals.

FIG. 3. (Left) Immunoblot analyses of c-PKC (A) and PKC� (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 �g of whole-celllysate protein, and the blot was developed using either a monoclonal antibody to different isotypes of c-PKC (A) or a monoclonal antibody to PKC�(B). Results obtained with a monoclonal antibody to the constitutively expressed protein �-actin are also shown (C) for comparison. (Right) Bargraphs present densitometric analysis results as ratios of intensities of PKC isoforms to those of �-actin bands. Results shown are representativeof three independent sets of experiments involving six animals.



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fact that the distribution and translocation of PKC isotypesremained largely unaffected (46). Although the exact mecha-nism underlying the observed attenuation of PKC activity orPKC-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 hostmacrophages (17, 23, 34). Since these inhibitors act either bydirect interaction (17, 34) with the host PKC inside the in-fected macrophages or indirectly by binding to their plasmamembranes (only after pretreatment of cells with high concen-trations of purified lipophosphoglycan) (23), they are less likelyto play a major role in the observed downregulation of PKCactivity in lymphocytes which do not harbor leishmanial para-sites.

Several studies indicate that phosphorylation of � and �isoforms of PKC in their activation loop is essential for theirpriming towards activation (16, 25). Further, membrane asso-ciation of PKC alone is now believed to be an insufficientcriterion for determination of its activity (56). In fact, thesestudies have emphasized the importance of dephosphorylationby phosphatases that act on PKC and thus modulate the dy-namics of the phosphorylation-dephosphorylation process incell physiology and function (40). The results presented in this

study appear to be consistent with this concept, as significantrestoration of PKC activity in lymphocytes is demonstrablefollowing pretreatment of SPMC and PBMC from infectedanimals with the Ser/Thr phosphatase inhibitor OA. Further,the presence of OA in the culture medium helped to achieveconsiderable restoration of lymphoproliferative responses ofSPMC and PBMC following stimulation with PMA plus Io. Wehave also seen marked suppression of lymphoproliferative re-sponses of SPMC and PBMC from infected hamsters to invitro stimulation with ConA, which could be partially, thoughsignificantly, restored following treatment with sodium or-thovanadate, 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 inthe signaling process of lymphocytes leading to their activationand proliferation. Leishmanial infection probably interfereswith this process by modulating these events through increasedintracellular activity of phosphatases. As a matter of fact, sev-eral studies have demonstrated that Leishmania-infected mu-rine macrophages exhibit elevated levels of PTPase activity(18, 33, 44), which may be responsible for downregulation ofprotein kinases and subsequent signal transduction processes.Interestingly, we have also noted higher levels of PTPase ac-tivity in lymphocytes derived from L. donovani-infected ham-sters than in their normal counterparts (Mookerjee and Ghose,unpublished).

Progressive visceral infection with L. donovani in hamstershas been shown to induce synthesis of the deactivating cyto-FIG. 5. (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 pre-sent densitometric analyses of the MAPK bands. Results are representa-tive of two independent sets of experiments involving four animals.

FIG. 6. Effects of in vitro OA or anti-TGF-� treatment of SPMCand PBMC from 45-day-infected hamsters on restoration of PKC ac-tivity in the membrane fractions of their nonadherent cell populations.PKC activities in the membrane fractions of nonadherent SPMC andPBMC from normal hamsters are also presented for the sake of com-parison. Results are means standard errors from three independentsets of experiments.



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kines such as interleukin-10 and TGF-� (35, 36). Further, theproliferative response of lymph node cells from hamsters im-munized with leishmanial antigen could be inhibited by ad-herent cells obtained from spleens of L. donovani-infectedhamsters, and such an inhibitory effect was abolished by ananti-TGF-� antibody (49). Increased synthesis of TGF-� inhamster VL has also been observed (P. Mukherjee, A. Mook-erjee, and A. C. Ghose, unpublished data). Furthermore, wealso observed downregulation of the lymphoproliferative re-sponse as well as the PKC activity of normal lymphocytes bysupernatants of overnight cultures of adherent cells from in-fected animals (data not shown). Significant, though partial,restoration of the lymphoproliferative response as well as ofPKC activity by an anti-TGF-� neutralizing antibody testifiesto the importance of this deactivating cytokine for the impair-ment of PKC activity and the manifestation of immunosup-pression in hamster VL. The importance of TGF-� in thesuppression of cellular immunity and the subsequent increasein susceptibility to infection has recently been established inmurine VL (58).

Several reports have demonstrated that TGF-� can act as asuppressor of cellular proliferation (5, 8, 21), which may beattributed to its ability to increase protein phosphatase activi-ties of target cells (8, 11, 21). Further, inhibition of cell cycleprogression by the TGF-� signaling pathway has been shownto be mediated through downregulation of cyclin A promoteractivity, which can be reversed by OA (59). Our results suggestthat a similar mechanism may be involved in hamster VL, asimpairment of the lymphoproliferative response in vitro maybe related to increased protein phosphatase activities, whichwould downregulate intracellular protein kinase activities. Thus,removal of adherent cells (the principal source of TGF-�)from the SPMC and PBMC helps in the partial restoration ofboth PKC activity and proliferation of lymphocytes from in-fected animals. Our recent data also indicate the importance ofTGF-� for impairment of PKC activity as well as for impair-ment of the proliferative response of lymph node cells derivedfrom hamsters with high levels of L. donovani infection (Mook-erjee and Ghose, unpublished). Further studies are under wayto ascertain the status of other protein kinases and phospha-tases in the immunosuppression associated with VL.

ACKNOWLEDGMENTS

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

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

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