The combination of apoptotic U937 cells and lupus IgG is a potent IFN-alpha inducer

Patients with active systemic lupus erythematosus (SLE) have signs of an ongoing IFN-alpha production, that may be of pathogenic significance in the disease. We previously showed that SLE patients have an IFN-alpha-inducing factor in blood, probably consisting of complexes containing anti-DNA Abs and immunostimulatory DNA. The DNA component could be derived from apoptotic cells, because SLE patients have been reported to have both increased apoptosis and reduced clearance of apoptotic cell material. In the present study, we therefore investigated whether apoptotic cells, together with IgG from SLE patients, could act as an IFN-alpha inducer in normal PBMC in vitro. We found that apoptotic cells of the myeloid leukemia cell line U937 as well as four other cell lines (MonoMac6, H9, Jurkat, U266) could induce IFN-alpha production in PBMC when combined with IgG from SLE patients. The IFN-alpha production by PBMC was much enhanced when PBMC were costimulated by IFN-alpha2b. The ability of IgG from different SLE patients to promote IFN-alpha induction by apoptotic U937 cells was associated with the presence of anti-ribonucleoprotein Abs, but not clearly with occurrence of anti-DNA Abs. These results suggest that apoptotic cells in the presence of autoantibodies can cause production of a clearly immunostimulatory cytokine, which is IFN-alpha. This mechanism for induction of IFN-alpha production could well be operative also in vivo, explain the IFN-alpha production seen in SLE patients, and be important in the pathogenesis of SLE.     

Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG

An ongoing production of IFN-alpha may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcgammaR in the IFN-alpha production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-alpha production, because Fab fragments or F(ab')(2) were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-alpha production, suggesting a role for FcgammaR on PBMC. Using blocking anti-FcgammaR Abs, the FcgammaRIIa,c (CD32) but not FcgammaRI or FcgammaRIII were shown to be involved in the IFN-alpha induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcgammaRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that approximately 50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcgammaRII, as did most of the actual IFN-alpha producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcgammaRIIa, but not of FcgammaRIIb or FcgammaRIIc. We conclude that FcgammaRIIa on NIPC/PDC is involved in the activation of IFN-alpha production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcgammaRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.     

Autoimmunity to DNA and nucleosomes in binary tetracycline-regulated polyomavirus T-Ag transgenic mice

The mechanism(s) responsible for autoimmunity to DNA and nucleosomes in SLE is largely unknown. We have demonstrated that nucleosome-polyomavirus T-Ag complexes, formed in context of productive polyomavirus infection, activate dsDNA-specific B cells and nucleosome-specific CD4(+) T cells. To investigate whether de novo expressed T-Ag is able to terminate nucleosome-specific T cell tolerance and to maintain anti-dsDNA Ab production in nonautoimmune mice, we developed two binary transgenic mouse variants in which expression of SV40 large T-Ag is controlled by tetracycline, MUP tTA/T-Ag (tet-off), and CMV rtTA/T-Ag (tet-on) mice. Data demonstrate that MUP tTA/T-Ag mice, but not CMV rtTA/T-Ag mice, are tightly controlling T-Ag expression. In MUP tTA/T-Ag transgenic mice, postnatal T-Ag expression activated CD8(+) T cells but not DNA-specific B cells, while immunization with T-Ag and nucleosome-T-Ag-complexes before T-Ag expression resulted in elevated and remarkably stable titers of anti-T-Ag and anti-dsDNA Abs and activation of T-Ag-specific CD4(+) T cells. Immunization of nonexpressing MUP tTA/T-Ag mice resulted in transient anti-T-Ag and anti-dsDNA Abs. This system reveals that a de novo expressed DNA-binding quasi-autoantigen maintain anti-dsDNA Abs and CD4(+) T cell activation once initiated by immunization, demonstrating direct impact of a single in vivo expressed molecule on sustained autoimmunity to DNA and nucleosomes.     

FCgammaRII (CD32)-dependent induction of interferon-alpha by serum from patients with lupus erythematosus

Interferon-alpha (IFN-alpha) is detected in the serum of 70-80% of patients with systemic lupus erythematosus (SLE). Furthermore, soluble factors in SLE serum can induce peripheral blood mononuclear cells (PBMC) to produce IFN-alpha. The purpose of this work was to investigate the mechanism of this IFN-alpha induction. In eleven of fifteen SLE serum samples, an IFN-alpha inducing activity was detected, whereas serum from healthy controls, patients with other autoimmune disease and patients with viral infections were ineffective under the same conditions. After gel filtration of the serum, the inducing activity was found in the same fraction as IgG. The IFN-alpha inducing activity was inhibited by native monoclonal antibodies to the receptors for the Fc portion of IgG: FcgammaRIIA/C and FcgammaRIIB subclasses (CD32) and by their F(ab)'2 fragments. Purified Fc fragments of human IgG were also effective in abolishing the IFN-alpha-inducing activity. Since no anti-CD32 autoantibodies were found in SLE serum, this IFN-alpha-inducing activity may be due to immune complex antibodies. Such results may allow better understand the origin of endogenous IFN-alpha, which has a deleterious effect on the course of this autoimmune disease. The inhibition of this function by the CD32 antibody could lead to new therapeutic approach in SLE.     

Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation of IFN-alpha production

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.     

Role of TLR9 in anti-nucleosome and anti-DNA antibody production in lpr mutation-induced murine lupus

Systemic lupus erythematosus is characterized by the production of autoantibodies directed against nuclear Ags, including nucleosome and DNA. TLR9 is thought to play a role in the production of these autoantibodies through the capacity of nuclear immunogenic particles to interact both with BCR and TLR9. To determine the role of TLR9 in SLE, C57BL/6-lpr/lpr-TLR9(-/-) and TLR9(+/+) mice were analyzed. The abrogation of TLR9 totally impaired the production of anti-nucleosome Abs, whereas no difference was observed in the frequency of anti-dsDNA autoantibodies whose titer was strikingly higher in TLR9(-/-) mice. In addition a higher rate of mesangial proliferation was observed in the kidney of TLR9-deficient animals. These results indicate that in C57BL/6-lpr/lpr mice, TLR9 is absolutely required for the anti-nucleosome Ab response but not for anti-dsDNA Ab production which is involved in mesangial proliferation.     

A distinct population of nonphagocytic and low level CD4+ null lymphocytes produce IFN-alpha after stimulation by herpes simplex virus-infected cells.

Human PBMC were stimulated for 6 h in vitro by HSV or Sendai virus (SV) and analyzed by flow cytometry. IFN-alpha producing cells (IPC) were identified through their content of IFN-alpha mRNA by in situ hybridization using a 35S-labeled IFN-alpha 2 cRNA probe. The IPC induced by HSV-infected WISH cells lacked capacity to adhere to and phagocytose latex particles. The induction of IFN-alpha by free infectious SV occurring in monocytes was abolished by phagocytosis of latex particles present in the cultures during the induction period. Such latex particles actually enhanced the IFN-alpha response induced by glutaraldehyde-fixed HSV- or SV-infected WISH cells or by free intact HSV. The HSV-induced IPC did not express the CD14 Ag expressed on monocytes. Cell sorting was performed on HSV-induced PBMC labeled with phycoerythrin-conjugated anti-CD3 and FITC-conjugated anti-CD4 mAb. A small population consisting of 1.4% of all PBMC, which was CD3- but expressed low but significant levels of CD4, contained the majority of the IPC with a 50-fold increase of their frequency. This cell population had a forward- and right-angle light scatter different from typical monocytes/macrophages. The results therefore further delineate IPC among PBMC into monocytes, being stimulated by viruses such as SV. Another distinct population of infrequent but highly efficient IPC, tentatively designated natural IFN-alpha producing cells, is activated by stimuli such as HSV.     

Novel roles for Lyn in B cell migration and lipopolysaccharide responsiveness revealed using anti-double-stranded DNA Ig transgenic mice

Lyn-deficient mice produce Abs against dsDNA, yet exhibit exaggerated tolerance to the model Ag hen-egg lysozyme. To investigate this apparent contradiction, and to further examine the function of Lyn in Ag-engaged cells, we have used an anti-dsDNA Ig transgenic model. Previously, looking at these anti-dsDNA B cells in Lyn-sufficient BALB/c mice, we showed that they are regulated by functional inactivation (anergy). In the absence of Lyn, these anti-dsDNA B cells remain unable to secrete Ab. This suggests that functional inactivation of anti-dsDNA B cells does not depend on Lyn, and that the anti-dsDNA Abs that are produced in lyn(-/-) mice arise from a defect in another mechanism of B cell tolerance. Although the anti-dsDNA B cells remain anergic, Lyn deficiency does restore their ability to proliferate to LPS. This reveals a novel role for Lyn in mediating the LPS unresponsiveness that normally follows surface Ig engagement. Furthermore, Lyn deficiency leads to an altered splenic localization and EBV-induced molecule 1 ligand chemokine responsiveness of anti-dsDNA B cells, as well as an absence of marginal zone B cells, suggesting additional roles for Lyn in controlling the migration and development of specific B cell populations.     

Alpha interferon inhibits human herpesvirus 8 (HHV-8) reactivation in primary effusion lymphoma cells and reduces HHV-8 load in cultured peripheral blood mononuclear cells

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.     

Autoimmune disease risk variant of IFIH1 is associated with increased sensitivity to IFN-α and serologic autoimmunity in lupus patients

Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease-associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α-induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA-positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 T allele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.     

Development of functional B cells in a line of SCID mice with transgenes coding for anti-double-stranded DNA antibody

Deletion or inactivation of anti-self (DNA) B cells has been reported in non-autoimmune mice bearing Ig transgenes that code for Abs with specificity for dsDNA or ssDNA. However, we report a case in which anti-dsDNA B cells appear to escape both deletion and inactivation. We show that B cells (B220+IgM+) can develop in non-autoimmune SCID mice bearing two site-directed transgenes, 3H9(56R) and Vkappa8, that together code for an anti-dsDNA Ab. The B cells appear inactive, because the mice (56RVkappa8 SCID mice) generally lack serum Ig. However, 56RVkappa8 SCID mice are able to produce IgG Ab with specificity for dsDNA when they become "leaky" for T cells or are reconstituted with exogenous T cells from B cell-deficient JH-/- donors. Thus, anti-dsDNA B cells that escape deletion in 56RVkappa8 SCID mice appear fully functional and can differentiate, class switch, and give rise to IgG-producing cells in the presence of T cells and self-Ag.     

Novel Autoantibodies Related to Cell Death and DNA Repair Pathways in Systemic Lupus Erythematosus

Systemic lupus erythematosus(SLE) is a complex autoimmune syndrome characterized by various co-existing autoantibodies(auto Abs) in patients' blood.However,the full spectrum of auto Abs in SLE has not been comprehensively elucidated.In this study,a commercial platform bearing 9400 antigens(Proto Array) was used to identify auto Abs that were significantly elevated in the sera of SLE patients.By comparing the auto Ab profiles of SLE patients with those of healthy controls,we identified 437 Ig G and 1213 Ig M auto Abs that the expression levels were significantly increased in SLE(P 0.05).Use of the Proto Array platform uncovered over 300 novel auto Abs targeting a broad range of nuclear,cytoplasmic,and membrane antigens.Molecular interaction network analysis revealed that the antigens targeted by the auto Abs were most significantly enriched in cell death,cell cycle,and DNA repair pathways.A group of auto Abs associated with cell apoptosis and DNA repair function,including those targeting APEX1,AURKA,POLB,AGO1,HMGB1,IFIT5,MAPKAPK3,PADI4,RGS3,SRP19,UBE2 S,and VRK1,were further validated by ELISA and Western blot in a larger cohort.In addition,the levels of auto Abs against APEX1,HMGB1,VRK1,AURKA,PADI4,and SRP19 were positively correlated with the level of anti-ds DNA in SLE patients.Comprehensive auto Ab screening has identified novel auto Abs,which may shed light on potential pathogenic pathways leading to lupus.     

Differential replication of human immunodeficiency virus type 1 in CD8- and CD8+ subsets of natural killer cells: relationship to cytokine production pattern.

CD8+ and CD8- subsets of peripheral blood natural killer (NK) cells were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1) and for the ability to produce various types of interferon (IFN) and tumor necrosis factor (TNF). HIV-1 was preferentially grown in CD8+ NK cells. The ability of CD8- NK cells to suppress HIV-1 replication was related to their ability to produce alpha IFN (IFN-alpha) upon viral induction. Induction with interleukin-2 resulted in IFN-gamma production in both subsets of NK cells. In the CD8+ subset, IFN-gamma and HIV-1 mutually enhanced the production of TNF alpha, leading to hyperactivation of viral replication, whereas in CD8- NK cells IFN-gamma primed HIV-induced IFN-alpha production. The dichotomous effects of IFN-gamma on HIV-1 replication were dependent on the IFN-alpha-producing ability of the cellular targets. These findings can explain the selective depletion of the CD16+ CD8+ subset that begins early in the in vivo HIV-1 infection.     

Induction of CD83+CD14+ nondendritic antigen-presenting cells by exposure of monocytes to IFN-alpha

IFN-alpha is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-alpha effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-alpha levels during treatment of infections and cancers. We show that in vitro IFN-alpha exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-alpha induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-alpha concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-alpha-challenged CD83(+) cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-alpha-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-alpha-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4(+) T cells. Notably, autologous memory CD4(+) T cells proliferated when exposed to tetanus toxoid-pulsed IFN-alpha-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-alpha showed long-lasting STAT-1 phosphorylation. Remarkably, CD83(+)CD14(+) cells were present in varicella skin lesions in close contact with IFN-alpha-producing cells. The present findings suggest that IFN-alpha alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-alpha in vivo.     

Antibody-mediated coengagement of FcγRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.     

Complex effects of interferon-alpha on the cytokine network in HIV infection--possible contribution to immunosuppression

Since interleukin (IL-)2, IL-10 and IL-12 may contribute to the pathogenesis of human immune deficiency virus (HIV) infection we examined the effect of interferon (IFN)-alpha on these cytokines in cultures of various subsets of peripheral blood mononuclear cells (PBMC) in ten HIV-infected patients and ten healthy controls. Our main findings were: (1) IFN-alpha markedly enhanced IL-10 levels in a dose-dependent manner in both lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-stimulated PBMC, as well as in anti-CD3- and anti-CD3/anti-CD28-stimulated T cells in both HIV-infected patients and controls. (2) In contrast, IFN-alpha had a downregulatory effect on IL-10 levels in Candida -stimulated PBMC,with particularly strong suppressive effect in HIV-infected patients. (3) Furthermore, IFN-alpha had a significant but modest stimulatory effect on IL-2 levels in PHA- and Candida -stimulated PBMC and anti-CD3-stimulated T cells. (4) IFN-alpha enhanced IL-12 levels in a dose-dependent manner in LPS-stimulated PBMC in both patients and controls. Our findings that IFN-alpha markedly enhanced IL-10 and modestly enhanced IL-2 and IL-12, suggest a net immunosuppressive effect of IFN-alpha in HIV-infected patients, possibly contributing to progression of immunodeficiency in these patients.     

Hepatitis C virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-alpha and plasmacytoid dendritic cell loss in chronic HCV infection

IFN-alpha production by plasmacytoid dendritic cells (PDCs) is critical in antiviral immunity. In the present study, we evaluated the IFN-alpha-producing capacity of PDCs of patients with chronic hepatitis C virus (HCV) infection in treatment-naive, sustained responder, and nonresponder patients. IFN-alpha production was tested in PBMCs or isolated PDCs after TLR9 stimulation. Treatment-naive patients with chronic HCV infection had reduced frequency of circulating PDCs due to increased apoptosis and showed diminished IFN-alpha production after stimulation with TLR9 ligands. These PDC defects correlated with the presence of HCV and were in contrast with normal PDC functions of sustained responders. HCV core protein, which was detectable in the plasma of infected patients, reduced TLR9-triggered IFN-alpha and increased TNF-alpha and IL-10 production in PBMCs but not in isolated PDCs, suggesting HCV core induced PDC defects. Indeed, addition of rTNF-alpha and IL-10 induced apoptosis and inhibited IFN-alpha production in PDCs. Neutralization of TNF-alpha and/or IL-10 prevented HCV core-induced inhibition of IFN-alpha production. We identified CD14+ monocytes as the source of TNF-alpha and IL-10 in the HCV core-induced inhibition of PDC IFN-alpha production. Anti-TLR2-, not anti-TLR4-, blocking Ab prevented the HCV core-induced inhibition of IFN-alpha production. In conclusion, our results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV core protein to induce cytokines that in turn lead to PDC apoptosis and inhibit IFN-alpha production. These mechanisms are likely to contribute to HCV viral escape from immune responses.     

Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis

Elevated spontaneous IgG production is characteristic of SLE. To identify the factors that support it, IL-6, a cytokine with an important role in the differentiation of IgG-secreting cells, was studied in SLE patients. Higher than normal levels of IL-6 were found, by a B9 assay, in sera of 63 of 70 patients (p less than 0.05). IL-6 was detected in 36 of 37 active SLE sera in higher titers (p = 0.009) than those for inactive SLE (n = 33), which were higher (p less than 0.05) than healthy controls (n = 15). IL-6 mRNA was detected in freshly isolated PBMC of 11 of 11 patients but not in normal PBMC, whereas IL-1 mRNA was detected only in patients with active disease. IL-6 activity was recovered from PBMC of four SLE patients, but not from four normal donors. By immunoperoxidase, IL-6 was detected in the cytoplasm of SLE monocytes and lymphocytes. When SLE PBMC were grown in short term cultures with no deliberate stimulation, expression of the IL-6 gene declined rapidly. Accordingly, the spontaneous production of IgG by SLE PBMC could be enhanced by exogenous IL-6. Spontaneous IgG production was diminished by 20 to 65% in the presence of neutralizing antibodies to IL-6, TNF-alpha, or IL-1. In contrast, neutralization of endogenous IL-4 increased production by approximately 40%. Anti-TNF-alpha treatment decreased IL-6 content of PBMC cultures, whereas anti-IL-4 augmented it, and exogenous IL-6 reversed anti-TNF-alpha effects on IgG production. Therefore, it is possible that the neutralization of TNF-alpha and IL-4 affected IgG production by modulating the synthesis/activity of IL-6. These results support the concept that SLE B cell hyperactivity is promoted by dysregulation of endogenous cytokines and suggest that IL-6, in particular, has an important pathogenic role.