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. 1997 Apr 1;94(7):3178-82.
doi: 10.1073/pnas.94.7.3178.

Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway

E Brigino  1 , S Haraguchi, A Koutsonikolis, G J Cianciolo, U Owens, R A Good, N K Day
Affiliations

Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway

E Brigino et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 Nef protein shares a significant homology with the immunosuppressive and highly conserved retroviral transmembrane protein p15E. In the present study, extracellular Nef protein is shown to induce interleukin (IL)-10 mRNA expression in human peripheral blood mononuclear cells as well as in cells of H9 T and U937 promonocytic human cell lines. Release of IL-10 protein into supernatants of peripheral blood mononuclear cells stimulated with Nef is dose-dependent. Expression of cytokines IL-2, IL-4, IL-5, IL-12 p40, IL-13, and interferon gamma is not affected by Nef stimulation. IL-10 protein production induced by Nef is inhibited by the calcium/calmodulin phosphodiesterase inhibitor W-7 but not by the protein kinase A inhibitor H-89 nor the protein kinase C inhibitors staurosporine and calphostin C. The calcium chelating agent EGTA also inhibits the IL-10 production induced by Nef, and this inhibition is reversed by the addition of calcium along with Nef. These findings indicate that extracellular Nef may contribute to the immunopathogenesis of HIV infection by inducing IL-10.

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Figures

Figure 1
Figure 1
RT-PCR analysis of the effects of E. coli-derived Nef on cytokine mRNA accumulation using human PBMCs. Human PBMCs were incubated for 3 h with medium (lane 1), 100 ng/ml staphylococcal enterotoxin A alone (lane 2), or 50 ng/ml Nef alone (lane 3).
Figure 2
Figure 2
Northern blot analysis of the effect of of E. coli-derived Nef on IL-10 mRNA expression using human PBMCs. PBMCs were incubated for 24 h with medium, 50 ng/ml Nef alone, or with 100 ng/ml staphylococcal enterotoxin A alone (positive control). Densitometric analysis was performed by reading the autoradiographic negative film with the Multiscan-R. IL-10 mRNA levels were normalized to β-actin mRNA levels. Arbitrary optical density units (AODU) were: medium = 0.45, Nef = 1.33, and staphylococcal enterotoxin A = 1.33.
Figure 3
Figure 3
Protein production of IL-10 in supernatants of PBMCs stimulated with increasing doses of yeast-derived Nef for 48 h. ELISA values represented are for pooled culture supernatants from five wells. IL-10 protein levels for those stimulated with 0, 10 (0.37 nM), 100, 500, and 1000 ng/ml of yeast-derived Nef are 7, 40, 49, 253, and 251 pg/ml, respectively. Similar dose-dependent influences of Nef derived from E. coli are also observed.
Figure 4
Figure 4
Effects of E. coli-derived Nef on IL-10 mRNA expression in H9 and U937 cells. (A) Northern blot analysis of the effect of E. coli-derived Nef on IL-10 mRNA expression in H9 cells after 2, 6, and 18 h of incubation. IL-10 mRNA expression with medium at 6 h of incubation is shown in the left lane. Densitometric analysis reveals the following arbitrary optical density units (AODU): 6 h medium = 0.547, 2 h Nef = 0.534, 6 h Nef = 3.303, and 18 h Nef = 0.881. (B) Northern blot analysis of the effect of E. coli-derived Nef on IL-10 mRNA expression using U937 cells. U937 cells pretreated with 1.2% dimethyl sulfoxide for 24 h were incubated with medium, 100 ng/ml Nef, or 50 ng/ml phorbol myristate acetate (positive control) for 3 h. AODU were: medium = 0.199, Nef = 0.399, and phorbol myristate acetate = 0.784.
Figure 5
Figure 5
Signal transduction experiments of IL-10 induction by Nef using several inhibitors. PBMCs were pretreated separately for 2 h with medium or inhibitor alone or were additionally stimulated with 500 ng/ml yeast-derived Nef alone or with 1.4 mM calcium chloride for 40 h. IL-10 production in the supernatants of PBMC from two different individuals was detected by ELISA. In Exp. 1, inhibitors used were 50 μM W-7, 1.5 nM staurosporine, and 100 nM H-89. In Exp. 2, inhibitors used were 50 μM W-7, 1.5 nM staurosporine, 100 nM H-89, 100 nM calphostin C, and 1 mM EGTA.
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