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 beta antibody

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 (Ca(2+), 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 beta (anti-TGF-beta) neutralizing antibody. These results correlated well with the ability of OA or the anti-TGF-beta 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-beta, levels of which are known to be elevated in visceral leishmaniasis.

FIG. 1.

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.

FIG. 2.

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.

FIG. 3.

(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.

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.

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 present densitometric analyses of the MAPK bands. Results are representative of two independent sets of experiments involving four animals.

FIG. 6.

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.

FIG. 7.

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.