Supernatants from HIV-1 infected macrophages and macrophages expressing HIV-1 Nef induce B cell proliferation, differentiation and immunoglobulin production. (A) Induction of B cell proliferation by HIV-1 Nef-expressing macrophage supernatants. Supernatants from macrophages 16 hrs post infection with recombinant Adenovirus expressing HIV-1 Nef or GFP (Swingler et al., 1999) or mock-infected were incubated with peripheral blood lymphocytes (left panel), purified B cells or T cells (middle panels). Tritiated thymidine incorporation was determined after 3 days and ³H-thymidine incorporation by B cells treated with a potent differentiation stimulus (αIgD/CD40L/IL-4) is shown for comparison (right panel) [error bars, SD]. (B-D) Supernatants from Nef-expressing macrophages and HIV-infected macrophages promote differentiation of B lymphocytes, ultimately to Plasma cells. Purified B cells were incubated with Nef-expressing (B) or HIV-1-infected (C) macrophage supernatants. Percent positive CD19, CD38 and CD70 cells are shown in bar charts for (B) and (C) [Error bars, SD]. FACS analysis (D) of surface CD138 and sIgM antigens was performed on CD38⁺-gated B cells following exposure to supernatants from Nef-expressing macrophages (upper panels) and HIV-1 infected (lower panels) macrophage supernatants. Percent positive cells are shown in each quadrant of the scatter plots. CD138 and sIgM expression on CD38⁺ gated cells exposed to a control stimulus (αIgD/IL-4/rCD40L) is shown for comparison (right hand panel). (E) B cell cultures exposed to supernatants from macrophages-expressing Nef secrete immunoglobulins. Purified B cells were treated with macrophage supernatants as in (A) and immunoglobulin levels in B cell supernatants were determined after 7 days. (F) HIV-1 WT infected macrophages drive immunoglobulin production from B cells. Supernatants from HIV-1 WT-infected, HIV-1 ΔNef-infected, and mock-infected macrophages were harvested at the peak of viral replication, added to purified B cells as in (E) and immunoglobulin levels determined after 7 days.

HIV-1 Nef induces ferritin production by macrophages but not T cells. (A) A high molecular weight factor in Nef-expressing macrophage supernatants induces B cell proliferation. Retentates (R) or filtrates (F), obtained following centrifugation of serum-free, Nef-expressing macrophage supernatant through various molecular weight cut-off filters, were added to purified B cells for analysis of proliferative activity (error bars, SD). (B) Micro-sequencing of a high molecular weight species from non-denaturing gels of concentrated Nef-expressing macrophage supernatant identified ferritin (upper panel). Accession numbers are 0901237A, NP000137, PO2792, and AAH69538. Nef induced ferritin (lower panel) comprises of two subunits supernatants that co-migrate with recombinant ferritin light chain (FTL) and heavy chain (FTH). (C) Nef-dependent induction of ferritin release from macrophages. Ferritin concentrations are shown in supernatants of macrophages expressing HIV-1 Nef or GFP and mock-infected macrophages. (D) Statistical analysis of ferritin release from macrophage cultures expressing Nef, GFP or mock (ANOVA; error bars, SEM). (E) Macrophages replicating HIV-1 containing an intact nef gene produce ferritin. At various intervals post-infection, supernatants were harvested from HIV-1 WT-infected, HIV-1 ΔNef-infected, and mock-infected macrophage cultures and analyzed for viral reverse transcriptase activity (right hand panel) and ferritin (left hand panel). (F) Statistical analysis of peak ferritin release from macrophage cultures infected with HIV-1 WT, HIV-1 ΔNef or mock (ANOVA; error bars, SEM). (G) CD4⁺ T lymphocytes do not produce ferritin in response to HIV-1 infection. CD3/CD28 activated CD4⁺ T cells or non-stimulated (quiescent) CD4⁺ T cells were infected with wild type (WT) or ΔNef HIV-1 variants or were left uninfected (mock). Cultures were monitored for viral replication (reverse transcriptase activity in culture supernatants; left panel) and for ferritin release (right panel). (H) HIV-1 infected macrophages do not release ferritin in response to paracrine factors. Supernatants from wild type and ΔNef HIV-1 infected, mock-infected or TNFα-treated macrophages were added to fresh uninfected macrophages in the presence and absence of AZT (to prevent de novo infection by viruses in culture supernatants) and ferritin release was evaluated after 4 or 24 hours.

HIV-1 Nef induces ferritin production by macrophages through an NF-κB-dependent pathway. (A, B) Nef-dependent induction of IκBα (A) and NF-κB-p105 (B) expression in HIV-1 infected macrophages. (C, D) Nef-dependent activation of IκBα. Macrophage lysates obtained at the peak of viral replication (C) were examined by Western blotting with antibodies to IκBα (D, lower panel) and with an antibody specific to the active (phosphorylated) form of IκBα (D, upper panel). (E), HIV-1 Nef expression in macrophages is sufficient for induction of IκBα and NF-κB-p105 induction in macrophages. Quantitative RT-PCR was performed approximately 16 hours post-transduction with adenovirus vectors expressing Nef or GFP. Non-transduced (mock) macrophages served as a control. (F) Specific activation of IκBα activating kinases IKKα/β in macrophages expressing HIV-1 Nef. IKKα/β levels were determined by ^32-Pin vitro kinase assay and densitometry. (G) Ferritin induction by HIV-1 Nef is blocked by a specific inhibitor of IκBα phosphorylation. Macrophages were infected singly or co-infected with the indicated adenovirus vectors and assayed for ferritin after 16 hours. IκBαSR is a dominant negative variant of IκBα containing serine to alanine mutations at positions 32 and 36 which prevent IκBα phosphorylation and subsequent degradation. (H) Expression of Nef itself was not impacted by the presence of the dominant negative IκBα. (I) Inhibition of CD40-dependent chemokine production (MIP-1α) by IκBαSR. (J) HIV-1 Nef is sufficient to upregulate ferritin mRNAs in macrophages. Real-time PCR analysis using primers specific to either ferritin light (FTL) or heavy (FTH) subunits was performed on RNA from macrophages transduced with adenovirus vectors expression Nef, GFP or on RNA from mock-infected or TNFα-stimulated macrophages.

B cell proliferation and immunoglobulin production by supernatants from Nef-expressing macrophages requires the presence of ferritin. (A) B cell proliferation depends on the presence of ferritin. Supernatants from macrophages expressing HIV-1 Nef, GFP, or untreated macrophages were immunodepleted with ferritin-specific or isotype antibodies (left hand panel) then examined for induction of proliferation in cultures of purified B cells (right hand panel). (B) Ferritin induces the production of immunoglobulin from B cells. Supernatants from Nef-expressing, GFP-expressing or mock-treated macrophages were immunodepleted by ferritin-specific or isotype antibodies as in (A), and assayed for the efficiency of ferritin immunodepletion (left hand panel) and for their ability to induce immunoglobulin production from purified B cells (right hand panels). (C) Nef immunodepletion does not impact B cell stimulation by supernatants of Nef-expression macrophages. Supernatants of Nef or GFP-expressing macrophages or mock-infected macrophages were treated with three Nef monoclonal antibodies or isotype controls and assayed for the ability to induced B cell proliferation. (D) Supernatants from macrophages treated with recombinant HIV-1 Nef or MA proteins do not induce B cell proliferation. Macrophages were treated with the recombinant proteins over a 6 log range and subsequently assessed for ability to induce B cell proliferation.

(A) Induction of B cell differentiation and immunoglobulin production by recombinant L-ferritin (FTL), H-ferritin (FTH) and purified ferritin (PF). Purified B cells were incubated with 200 ng.ml⁻¹ of each ferritin (equivalent to the concentration induced by Nef-expressing supernatants) and cellular proliferation assessed by thymidine incorporation. Purified ferritin promotes differentiation of B lymphocytes (B and C). B cells were incubated with ferritin (200 ng.ml⁻¹) as in (A) and flow-cytometric analyses performed for CD19, CD38 and CD70 expression at five days (B) and for CD138 and sIgM antigens at 7 days post exposure (C). Percent positive cells are shown in bar charts for (B) [Error bars, SD] and in each quadrant of scatter plots for CD38⁺-gated B-cells (C). (D) Ferritin alone is sufficient to drive immunoglobulin production from purified B cells. Increasing concentrations of L-ferritin, H-ferritin or purified ferritin were incubated with cultures of B cells and examined for immunoglobulin production at 7 days post-exposure. (E) Induction of ERK and NF-κB pathways in B cells by purified ferritin. Fold increases in mRNA levels in B cells treated with purified ferritin (PF), mock-treated B cells or B cells subjected to an activation stimulus (αIgD/IL-4/rCD40L) are shown relative to levels in untreated (mock) B cells. Fold changes >1.7X are considered significantly different (Butte et al., 2001). (F) Activation of NF-κB and ERK pathways in B cells by purified ferritin. Lysates of B cells treated with purified ferritin (PF) or a B cell activation stimulus (αIgD/IL-4/rCD40L) were Western blotted with IκBα-specific antibodies or antibodies specific for active (phosphorylated) IκBα. ERK3 activation was assessed by ability of ERK3 immunoprecipitates to induce phosphorylation of myelin basic protein (MBP) in an in vitro kinase assay. (G) Effect of NF-κB and MEK inhibitors on B cell activation by ferritin. B cells were subject to the indicated stimuli in the presence and absence of the NF-κB inhibitor CAPE (5 μg ml⁻¹) or the MEK inhibitor PD98059 (10 μM). (H), comparative analysis of expression profiles of genes involved in transcription, B cell activation/proliferation and differentiation to immunoglobulin secreting plasma cells in B cells treated with purified ferritin, untreated cells or B cells exposed to an activation stimulus (αIgD/IL-4/rCD40L). Results are calculated as fold increase in mRNA levels relative to mock-treated B cells.

In a cohort of HIV-1 infected patients vRNA load correlates with ferritin, IgA, IgG and IgM levels in plasma. (A) Correlation between plasma HIV-1 RNA, immunoglobulin and ferritin levels in HIV-1-infected individuals (n=83) was examined by linear regression analysis where p indicates the significance of the relationship and r indicates the strength of the association between the parameters. Best-fit lines are indicated in red and grey dashed lines show the 95% confidence limits. (B) Ferritin and IgA, IgG and IgM levels correlate in HIV-1 infected individuals. (C) No significant relationship occurs between ferritin and plasma levels of the immune activation markers sTNFRII (left panel) and β-2 microglobulin (right panel). Scatter plots and statistical analyses for (B) and (C) were performed as in (A). (D) Analysis of HIV-1 infected and uninfected individuals demonstrates HIV-dependent hyperferritinemia and hypergammaglobulinemia (t-test; error bars, SEM).

HIV-1 Nef induces ferritin release from the murine macrophages and mice transgenic for HIV-1 Nef exhibit hyperferritinemia and hypergammaglobulinemia. (A) The murine macrophage cell line RAW 264.7 was transiently transfected with plasmids expressing HIV-1 Nef, CAT, mock-treated or stimulated with IFNγ/LPS and ferritin levels in supernatants measured after 16 hrs (B) Statistical analysis of ferritin release from murine macrophage cell cultures expressing HIV-1 Nef, CAT, mock-treated or stimulated with IFNγ/LPS (ANOVA; error bars, SEM). (C) Analysis of HIV-1 Nef transgenic mice and control non-Tg litter mates demonstrates statistically significant Nef-dependent increases in serum ferritin and immunoglobulins, IgA, IgG and IgM (t-test; error bars, SEM).