Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani

Abstract

Intracellular protozoan parasites of the genus Leishmania antagonize host defense mechanisms by interfering with cell signaling in macrophages. In this report, the impact of Leishmania donovani on mitogen-activated protein (MAP) kinases and nitric oxide synthase (NOS) expression in the macrophage cell line RAW 264 was investigated. Overnight infection of cells with leishmania led to a significant decrease in phorbol-12-myristate-13-acetate (PMA)-stimulated MAP kinase activity and inhibited PMA-induced phosphorylation of the MAP kinase substrate and transcription factor Elk-1. Simultaneously, leishmania infection markedly attenuated the induction of c-FOS and inducible nitric oxide synthase (iNOS) expression in response to PMA and gamma interferon (IFN-gamma), respectively. These effects correlated with decreased phosphorylation of p44 and p42 MAP kinases on tyrosine residues. Consistent with the latter finding, lysates prepared from leishmania-infected cells contained an activity that dephosphorylated MAP kinase in vitro, suggesting the possibility of a phosphatase acting in vivo. Attenuation of both MAP kinase activity and c-FOS and iNOS expression was reversed by treatment of macrophages with sodium orthovanadate prior to infection. It was also found that the specific activity of the Src homology 2 domain containing tyrosine phosphatase (SHP-1) toward MAP kinase was markedly increased in leishmania-infected cells. These findings indicate that infection with L. donovani attenuates MAP kinase signaling and c-FOS and iNOS expression in macrophages by activating cellular phosphotyrosine phosphatases. This may represent a novel mechanism of macrophage deactivation during intracellular infection.

FIG. 1

L. donovani attenuates PMA-induced tyrosine phosphorylation of MAP kinases (MAPKs). Cells were either untreated or incubated with leishmania amastigotes at an approximate parasite-to-cell ratio of 15:1. After overnight incubation (17 h), control and infected cells were incubated in the absence or presence of 100 nM PMA for 15 min. (A) Cells were lysed in modified RIPA buffer as described in Materials and Methods. Whole-cell lysates were separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide), transferred to nitrocellulose membranes, and probed with antiphosphotyrosine (anti PY) antibodies. Blots were developed by ECL, and an autoluminogram of a blot is shown. The tyrosine-phosphorylated bands with M_r of 44,000 and 42,000 corresponded to p44^{MAP kinase-1} and p42^{MAP kinase-2}, respectively. In addition to MAP kinase-1 and MAP kinase-2, the positions of other PMA-induced phosphotyrosine-containing proteins are indicated by arrows. The autoluminogram was analyzed by densitometry in the region of p44 and p42 MAP kinases. (B) The same blot was stripped and reprobed with anti-MAP kinase antibodies. The data shown are from three independent experiments that yielded similar results. The values shown in the histogram represent mean and standard deviation.

FIG. 2

L. donovani infection attenuates phosphotransferase activity of MAP kinase. Cells were infected with L. donovani at an approximate parasite-to-cell ratio of 15:1. After overnight incubation (17 h), control and infected cells were stimulated with 100 nM PMA (A) or LPS (1 μg/ml) (B) for 15 min. The cells were lysed and immunoprecipitated with anti-MAP kinase (anti MAPK 1) antibody. MBP phosphorylating activity was measured in an immune complex kinase assay as described in Materials and Methods. Phosphorylated MBP was electrophoresed by SDS-polyacrylamide gel electrophoresis (15% polyacrylamide) and transferred to a nitrocellulose membrane. After being stained with amido black, the blot was dried and exposed to X-ray film. After autoradiography, the blot was blocked and probed with anti-MAP kinase antibody to assess the input of precipitated MAP kinase. After immunoblotting, the bands in the region of MBP were excised and counted by liquid scintillation counting. (A) The data shown are from one of three independent experiments that yielded similar results. Values in the histogram represent mean and standard deviation. (B) Values in the histogram represent the average of two determinations from independent experiments.

FIG. 3

L. donovani attenuates serine phosphorylation of Elk-1. Cells were incubated with or without amastigotes of L. donovani for 17 h and then stimulated with 100 nM PMA for 15 min. Detergent lysates were prepared with modified RIPA buffer as described in Materials and Methods, and soluble proteins were separated by SDS-polyacrylamide gel electrophoresis (7.5% polyacrylamide) followed by transfer to nitrocellulose membranes. The membranes were probed with phospho-Ser³⁸³-specific Elk-1 antibody. The blots were developed by ECL, and an autoluminogram is shown. The data shown are representative of two independent experiments that gave similar results; values in the histogram represent the average.

FIG. 4

L. donovani infection stimulates an activity that dephosphorylates MEK-phosphorylated MAP kinase-1–GST. MAP kinase-1–GST (MAPK 1-GST) coupled to glutathione-agarose was phosphorylated with MEK and [γ-³²P]ATP in phosphorylation buffer as described in Materials and Methods. Phosphorylated MAP kinase-1–GST agarose beads were incubated with whole-cell lysates prepared from control and leishmania-infected (17 h) cells treated with or without 100 nM PMA. The phosphatase assay was stopped by the addition of SDS sample buffer. (A) The phosphotyrosine content in MAP kinase-1 in PMA-treated control and infected cells was analyzed by immunoblotting with anti-phosphotyrosine (anti PY) antibodies. The autoluminogram shown was analyzed by densitometry. (B) The same membrane was exposed to X-ray film to determine the ³²P content. (C) The membrane was stripped and probed with anti-MAP kinase antibody to assess the level of MAP kinase-1–GST input protein. The data shown are representative of three independent experiments that gave similar results; values in the histogram represent mean and standard deviation.

FIG. 5

Attenuation of MAP kinase activity in leishmania-infected cells is abrogated by sodium orthovanadate. Cells were preincubated with sodium orthovanadate (20 μM) for 3 h and then infected (17 h) with leishmania amastigotes at an approximate parasite-to-cell ratio of 15:1. Control and infected cells were then stimulated with PMA (100 nM) for 15 min. Cell lysates were immunoprecipitated with anti-MAP kinase antibody. MBP phosphorylation was assessed in an immune complex assay as described in Materials and Methods. Phosphorylated MBP was electrophoresed on SDS–15% polyacrylamide gels and electroblotted to nitrocellulose membranes. After being stained with amido black, the blots were dried and exposed to X-ray film. The bands in the region of MBP were excised and counted by liquid scintillation counting. The data shown are the mean and standard deviation of values obtained in three independent experiments.

FIG. 6

Pretreatment of cells with sodium orthovanadate reverses the effect of leishmania infection on c-FOS protein expression. Cells were either untreated or treated with orthovanadate (20 μM) for 3 h and then incubated with leishmania amastigotes at an approximate parasite-to-cell ratio of 15:1. After overnight incubation, control and infected cells were stimulated with PMA (100 nM) for 4 h. Cells were lysed in modified RIPA buffer as described in Materials and Methods. Whole-cell lysates were separated on SDS–7.5% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with c-FOS antibodies. The blots were developed by ECL, and an autoluminogram of a blot is shown. The blot shown is from one of two independent experiments that yielded similar results, and the histogram shows the average values of the two experiments.

FIG. 7

Pretreatment of cells with sodium orthovanadate abrogates the effect of leishmania infection on iNOS protein expression. Cells were either untreated or treated with orthovanadate (20 μM) for 3 h and then incubated with leishmania amastigotes at an approximate parasite-to-cell ratio of 15:1. After overnight incubation (17 h), control and infected cells were stimulated with IFN-γ for 6 h. Cells were lysed in modified RIPA buffer as described in Materials and Methods. Whole-cell lysates were separated on SDS–7.5% polyacrylamide gels, transferred to nitrocellulose membranes, and probed with anti-iNOS antibodies. The blots were developed by ECL, and an autoluminogram of a blot is shown. The data shown are from two independent experiments that yielded similar results, and values in the histogram represent the average.

FIG. 8

L. donovani infection increases the specific activity of SHP-1 without affecting either tyrosine phosphorylation status or cellular levels of enzyme. (A) Cells were infected with leishmania for 17 h. Control and infected cells were lysed in buffer as described in Materials and Methods. Detergent-solubilized proteins were immunoprecipitated with antibody to SHP-1. Immunoprecipitated SHP-1 was incubated with autophosphorylated MAP kinase-1–GST (as described in Materials and Methods) for 1 h at 30°C. Reactions were stopped by adding SDS sample buffer. Phosphatase activity was assessed by immunoblotting with antiphosphotyrosine (anti PY) antibodies. The autoluminogram shown was analyzed by densitometry. The blots were then stripped and reprobed with anti-SHP-1 to determine the level of immunoprecipitated SHP-1. The same membranes were again stripped and reprobed with anti-MAP kinase (anti MAPK 1) to assess the input of MAP kinase-1–GST. (B) (Top) Cells infected with leishmania (17 h) were lysed in buffer, immunoprecipitated with anti-SHP-1, and immunoblotted with antiphosphotyrosine antibodies to assess the tyrosine phosphorylation status of SHP-1. (Bottom) Cells infected with leishmania (17 h) were lysed in modified RIPA buffer, and solubilized proteins were separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide) followed by immunoblotting. Total cellular levels of SHP-1 were measured by immunoblotting with anti-SHP-1 antibody. The data shown in lanes with error bars are the mean and standard deviation of results obtained in three independent experiments. Other results are control results from one experiment.

FIG. 9

Time course of activation of SHP-1 by L. donovani. Cells were infected with leishmania for different time intervals as indicated, lysed in buffer without phosphatase inhibitors, and processed for immunoprecipitation of SHP-1. Immunoprecipitated SHP-1 was incubated with autophosphorylated MAP kinase-1–GST (MAPK 1-GST) for 1 h at 30°C as described in the legend to Fig. 5. Phosphatase activity was assessed by immunoblotting with antiphosphotyrosine (anti PY) antibody. The autoluminogram shown was analyzed by densitometry. The blot was then stripped and reprobed with anti-SHP-1 to assess the level of immunoprecipitated SHP-1. The same membrane was again stripped and probed with anti-MAP kinase to assess the input of MAP kinase-1–GST. The data shown are from one of two experiments that yielded similar results, and the values in the histogram represent the average.